# Patent

## Title
Non-endogenous, constitutively activated known G protein-coupled receptors

## Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to a human G protein-coupled receptor for which the endogenous ligand is known (“known GPCRs”), and most particularly to mutated (non-endogenous) versions of the known GPCRs for use, most preferably in screening assays for the direct identification of candidate compounds as inverse agonists, agonists and partial agonists.

## Background
The present application is a Continuation of U.S. Utility patent application Ser. No. 10/925,095, filed Aug. 24, 2004, issued as U.S. Pat. No. 7,097,969, which claims priority to Divisional of U.S. Utility patent application Ser. No. 09/826,509, filed Apr. 5, 2001, now U.S. Pat. No. 6,806,054, issued Oct. 19, 2004, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 60/195,747, filed with the United States Patent and Trademark Office on Apr. 7, 2000, each of which is hereby incorporated by reference.


FIELD OF THE INVENTION
The invention disclosed in this patent document relates to transmembrane receptors, and more particularly to G protein-coupled receptors for which the endogenous ligand has been identified (“known GPCR”), and specifically to known GPCRs that have been altered to establish or enhance constitutive activity of the receptor. Most preferably, the altered GPCRs are used for the direct identification of candidate compounds as receptor agonists, inverse agonists or partial agonists for use as therapeutic agents.
BACKGROUND OF THE INVENTION
Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR or GPCRs) class. It is estimated that there are some 100,000 genes within the human genome, and of these, approximately 2%, or 2,000 genes, are estimated to code for GPCRs. Receptors, including GPCRs, for which the endogenous ligand has been identified are referred to as “known” receptors, while receptors for which the endogenous ligand has not been identified are referred to as “orphan” receptors. GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, 60% of all prescription pharmaceuticals have been developed.
GPCRs share a common structural motif. (All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmebrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane (these are referred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane (these are referred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
Generally, when an endogenous ligand binds with the receptor (often referred to as “activation” of the receptor), there is a change in the conformation of the intracellular region that allows for coupling between the intracellular region and an intracellular “G-protein.” It has been reported that GPCRs are “promiscuous” with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to produce a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
A receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than endogenous ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of an endogenous ligand binding to the receptor. Stabilization by such ligand-independent means is termed “constitutive receptor activation.”
Traditional ligand-dependent screens seek to indirectly identify compounds that antagonize the action of the ligand on the receptor in an effort to prevent ligand-induced activation of the receptor. However, such compounds, sometimes referred to as neutral-antagonists, generally would not be expected to affect the ligand-independent activity, or overactivity, of the receptor and the subsequent abnormal cellular response that can result from this overactivity. This is particularly relevant to a growing number of diseases, such as those identified in the table below, that have been linked to overactive GPCRs, because traditional neutral-antagonists will not block the abnormal ligand-independent activity of these receptors.










Background Table 1










Disease
Overactive GPCR







Schizophrenia
5-HT2A, D2



Depression
5-HT2A



Hyperthyroidism
Thyrotropin



Hypertension
Angiotensin AT1A



Asthma
Adenosine A1



Melanoma
MC-1



Retinitis Pigmentosa
Rhodopsin receptor










SUMMARY OF THE INVENTION
Disclosed herein are non-endogenous versions of endogenous, known GPCRs and uses thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides graphic results of comparative analysis of a co-transfection of non-endogenous TSHR-A623I (“signal enhancer”) and an endogenous target receptor, in this case GPR24 (“GPR24 wt”), versus non-endogenous, constitutively activated versions of the target receptor GPR24 (“T255K,” “T255K/T257R,” “24-IC3-SST2,” “C305Y,” “P271L,” “W269C,” “W269F,” “W269L,” “F265I,” “I261Q,” and “D140N”) co-transfected with non-endogenous TSHR-A623I, utilizing an adenylyl cyclase assay. This assay involved the addition of TSH and MCH, the endogenous ligands for TSHR and GPR24, respectively.
FIG. 2 provides graphic results of comparative analysis of a co-transfection of non-endogenous signal enhancer TSHR-A623I (with and without TSH) and endogenous target receptor GPR5 (“GPR5 wt”), versus non-endogenous, constitutively activated target receptor GPR5 (“V224K”) co-transfected with non-endogenous TSHR-A623I (with and without TSH), utilizing an adenylyl cyclase assay.
FIGS. 3A-3E provide a diagrammatic representation of the signal measured comparing CMV, non-endogenous, constitutively activated GPCRs, utilizing 8×CRE-Luc reporter plasmid.
FIG. 4 provides an illustration of IP3 production from several non-endogenous versions of GPR24 as compared with the endogenous version of this receptor.
FIG. 5 is a graphic representation of the results of a membrane-based cyclic AMP assay providing comparative results for constitutive signaling of TSHR-A623K:Fusion Protein and a control vector (pCMV).



DETAILED DESCRIPTION
The scientific literature that has evolved around receptors has adopted a number of terms to refer to ligands having various effects on receptors. For clarity and consistency, the following definitions will be used throughout this patent document. To the extent that these definitions conflict with other definitions for these terms, the following definitions shall control:
AGONISTS shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor, or enhance GTP binding to membranes.
AMINO ACID ABBREVIATIONS used herein are set out in Table A:











TABLE A









ALANINE
ALA
A



ARGININE
ARG
R



ASPARAGINE
ASN
N



ASPARTIC ACID
ASP
D



CYSTEINE
CYS
C



GLUTAMIC ACID
GLU
E



GLUTAMINE
GLN
Q



GLYCINE
GLY
G



HISTIDINE
HIS
H



ISOLEUCINE
ILE
I



LEUCINE
LEU
L



LYSINE
LYS
K



METHIONINE
MET
M



PHENYLALANINE
PHE
F



PROLINE
PRO
P



SERINE
SER
S



THREONINE
THR
T



TRYPTOPHAN
TRP
W



TYROSINE
TYR
Y



VALINE
VAL
V










PARTIAL AGONISTS shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor to a lesser degree/extent than do agonists, or enhance GTP binding to membranes to a lesser degree/extent than do agonists.
ANTAGONIST shall mean materials (e.g., ligands, candidate compounds) that competitively bind to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses by agonists or partial agonists. ANTAGONISTS do not diminish the baseline intracellular response in the absence of an agonist or partial agonist.
CANDIDATE COMPOUND, in the context of the disclosed invention, shall mean a small molecule (for example, and not limitation, a chemical compound) that is amenable to a screening technique.
COMPOSITION means a material comprising at least one component; a “pharmaceutical composition” is an example of a composition.
COMPOUND EFFICACY shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality, as opposed to receptor binding affinity. Exemplary means of detecting compound efficacy are disclosed in the Example section of this patent document.
CODON shall mean a grouping of three nucleotides (or equivalents to nucleotides) which generally comprise a nucleoside (adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)) coupled to a phosphate group and which, when translated, encodes an amino acid.
CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subject to constitutive receptor activation. A constitutively activated receptor can be endogenous or non-endogenous.
CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of a receptor in the active state by means other than binding of the receptor with its endogenous ligand or a chemical equivalent thereof.
CONTACT or CONTACTING shall mean bringing at least two moieties together, whether in an in vitro system or an in vivo system.
DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship to the phrase “candidate compound”, shall mean the screening of an candidate compound against a constitutively activated receptor, preferably a constitutively activated receptor, and most preferably against a constitutively activated G protein-coupled cell surface receptor, and assessing the compound efficacy of such compound. This phrase is, under no circumstances, to be interpreted or understood to be encompassed by or to encompass the phrase “indirectly identifying” or “indirectly identified.”
ENDOGENOUS shall mean a material that a mammal naturally produces. ENDOGENOUS in reference to, for example and not limitation, the term “receptor,” shall mean that which is naturally produced by a mammal (for example, and not limitation, a human) or a virus. By contrast, the term NON-ENDOGENOUS in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) or a virus. For example, and not limitation, a receptor which is not constitutively active in its endogenous form, but when manipulated becomes constitutively active, is most preferably referred to herein as a “non-endogenous, constitutively activated receptor.” Both terms can be utilized to describe both “in vivo” and “in vitro” systems. For example, and not limitation, in a screening approach, the endogenous or non-endogenous receptor may be in reference to an in vitro screening system. As a further example and not limitation, where the genome of a mammal has been manipulated to include a non-endogenous constitutively activated receptor, screening of a candidate compound by means of an in vivo system is viable.
G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, in the context of the invention disclosed herein, each mean a non-endogenous protein comprising an endogenous, constitutively activate GPCR or a non-endogenous, constitutively activated GPCR fused to at least one G protein, most preferably the alpha (α) subunit of such G protein (this being the subunit that binds GTP), with the G protein preferably being of the same type as the G protein that naturally couples with endogenous GPCR. For example, and not limitation, in an endogenous state, if the G protein “Gsα” is the predominate G protein that couples with the GPCR, a GPCR Fusion Protein based upon the specific GPCR would be a non-endogenous protein comprising the GPCR fused to Gsα; in some circumstances, as will be set forth below, a non-predominant G protein can be fused to the GPCR. The G protein can be fused directly to the c-terminus of the constitutively active GPCR or there may be spacers between the two.
HOST CELL shall mean a cell capable of having a Plasmid and/or Vector incorporated therein. In the case of a prokaryotic Host Cell, a Plasmid is typically replicated as a autonomous molecule as the Host Cell replicates (generally, the Plasmid is thereafter isolated for introduction into a eukaryotic Host Cell); in the case of a eukaryotic Host Cell, a Plasmid is integrated into the cellular DNA of the Host Cell such that when the eukaryotic Host Cell replicates, the Plasmid replicates. Preferably, for the purposes of the invention disclosed herein, the Host Cell is eukaryotic, more preferably, mammalian, and most preferably selected from the group consisting of Hek-293, Hek-293T and COS-7 cells.
INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the traditional approach to the drug discovery process involving identification of an endogenous ligand specific for an endogenous receptor, screening of candidate compounds against the receptor for determination of those which interfere and/or compete with the ligand-receptor interaction, and assessing the efficacy of the compound for affecting at least one second messenger pathway associated with the activated receptor.
INHIBIT or INHIBITING, in relationship to the term “response” shall mean that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
INVERSE AGONISTS shall mean materials (e.g., ligand, candidate compounds) which bind to either the endogenous form of the receptor or to the constitutively activated form of the receptor, and which inhibit the baseline intracellular response initiated by the active form of the receptor below the normal base level of activity which is observed in the absence of agonists or partial agonists, or decrease GTP binding to membranes. Preferably, the baseline intracellular response is inhibited in the presence of the inverse agonist by at least 30%, more preferably by at least 50%, and most preferably by at least 75%, as compared with the baseline response in the absence of the inverse agonist.
KNOWN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has been identified.
LIGAND shall mean an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.
MUTANT or MUTATION in reference to an endogenous receptor's nucleic acid and/or amino acid sequence shall mean a specified change or changes to such endogenous sequences such that a mutated form of an endogenous, non-constitutively activated receptor evidences constitutive activation of the receptor. In terms of equivalents to specific sequences, a subsequent mutated form of a human receptor is considered to be equivalent to a first mutation of the human receptor if (a) the level of constitutive activation of the subsequent mutated form of a human receptor is substantially the same as that evidenced by the first mutation of the receptor; and (b) the percent sequence (amino acid and/or nucleic acid) homology between the subsequent mutated form of the receptor and the first mutation of the receptor is at least about 80%, more preferably at least about 90% and most preferably at least 95%. Ideally, and owing to the fact that the most preferred cassettes disclosed herein for achieving constitutive activation includes a single amino acid and/or codon change between the endogenous and the non-endogenous forms of the GPCR, the percent sequence homology should be at least 98%.
NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring molecule specific for an endogenous naturally occurring ligand wherein the binding of a ligand to a receptor activates an intracellular signaling pathway.
ORPHAN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has not been identified or is not known.
PHARMACEUTICAL COMPOSITION shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, and not limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.
PLASMID shall mean the combination of a Vector and cDNA. Generally, a Plasmid is introduced into a Host Cell for the purposes of replication and/or expression of the cDNA as a protein.
STIMULATE or STIMULATING, in relationship to the term “response” shall mean that a response is increased in the presence of a compound as opposed to in the absence of the compound.
VECTOR in reference to cDNA shall mean a circular DNA capable of incorporating at least one cDNA and capable of incorporation into a Host Cell.
The order of the following sections is set forth for presentational efficiency and is not intended, nor should be construed, as a limitation on the disclosure or the claims to follow.
A. Introduction
Constitutively active forms of known G protein-coupled receptors, disclosed in the present patent document, can be obtained by site-directed mutational methods, well-known to those skilled in the art. A constitutively active receptor useful for direct identification of candidate compounds is preferably achieved by mutating the receptor at a specific location within an intracellular loop, most preferably within the intracellular loop three (IC3) region. Such mutation can produce a non-endogenous receptor that is intended to be constitutively activated, as evidenced by an increase in the functional activity of the receptor, for example, an increase in the level of second messenger activity. While standard methods of site-directed mutagenesis may be employed, a preferred method is one that is disclosed in a co-pending, commonly assigned patent document U.S. application Ser. No. 09/170,496, incorporated herein by reference.
Table B below lists known endogenous GPCRs that have been converted to non-endogenous versions thereof, their respective G protein and endogenous ligand.









TABLE B





Known GPCRs
G Protein
Endogenous Ligand







5HT-1A
Gi
Serotonin


5HT-1B
Gi
Serotonin


5HT-1D
Gi
Serotonin


5HT-1E
Gi
Serotonin


5HT-1F
Gi
Serotonin


5HT-2B
Gi
Serotonin


5HT-4A
N/I
Serotonin


5HT-4B
N/I
Serotonin


5HT-4C
N/I
Serotonin


5HT-4D
N/I
Serotonin


5HT-4E
N/I
Serotonin


5HT-5A
Unknown
Serotonin


5HT-6
Gs
Serotonin


5HT-7
Gs
Serotonin


AVPR1A
Gq
Arginine Vasopressin


AVPRIB
Gq
Arginine Vasopressin


AVPR2
Gs
Arginine Vasopressin


BBR3
Gq
Bombesin


BDKR1
Gq
Bradykinin


BDKR2
Gq
Bradykinin


C3a
N/I
Anaphylatoxin


C5a
N/I
Anaphylatoxin


CB1
Gi
Cannabinoid


CB2
Gi
Cannabinoid


CCR2b
Gi
Monocyte chemoattractant




(MCP)


CCR3
Gi
Eotaxin, Leukotactin-1,




RANTES, MCP


CCR5
Gi
MIP-1α, MIP-1β,




RANTES


CCR8
Gi
I-309, TARC, MIP-1β


CCR9
N/I
Thymus-expressed




chemokine (TECK)


CRFR1
Gs
Corticotropin-releasing-




factor


CXCR4
Gi
SDF1


Dopamine D1
Gs
Dopamine


Dopamine D2
Gi
Dopamine


Dopamine D3
Gi
Dopamine


Dopamine D5
Gs
Dopamine


ETA
Gq
Endothelin


ETB
Gq
Endothelin


FPR1
N/I
Formylpeptide


FPRL1
N/I
formylpeptide


GALR1
Gi
Galanin


GALR2
Gi/Gq
Galanin


GIP
N/I
Gastric inhibitory




polypeptide


mGluR1
Gq
Glutamate


GPR5
N/I
Single C motif-1 (SMC-1)


GPR24 (also known as MCH or
Gi/Gq
Melanin Concentrating


SLC-1)

Hormone


GRPR
Gq
Gastrin releasing peptide


M1
Gq
Acetylcholine


M2
Gi
Acetylcholine


M3
Gq
Acetylcholine


M4
Gi
Acetylcholine


M5
Gq
Acetylcholine


MC3
Gs
Melanocortin


NK1R
Gq
Substance P


NK2R
Gq
Neurokinin-A


NK3R
Gq
Neurokinin-B


NMBR
Gq
Neuromedin B


NPY5
Gi
Neuropeptide Y


NTSR1
Gq
Neurotensin


NTSR2
Gq
Neurotensin


OPRD
Gi
Opiod


OPRL1
Gi
Opiod


OPRK
Gi
Opiod


OPRM
Gi
Opiod


OPRM1A
Gi
Opiod


OX1R
N/I
Orexin


OX2R
N/I
Orexin


PACAP
Gs
Pituitary adenylyl cyclase




activating peptide


PAF
N/I
Platelet activating factor


PGE EP1
N/I
Prostaglandin


PGE EP2
Gs
Prostaglandin


PGE EP4
N/I
Prostaglandin


PTHR1
N/I
Parathyroid hormone


PTHR2
N/I
Parathyroid hormone


SCTR
Gs
Secretin


SST1
Gi
Somatostatin


SST2
Gi
Somatostatin


SST3
Gi
Somatostatin


SST4
Gi
Somatostatin


SST5
Gi
Somatostatin


TSHR
Gs
Thyroid Stimulating




Hormone


VIPR
Gs
Vasoactive Intestinal




Peptide


VIPR2
Gs
Vasoactive Intestinal




Peptide





Note:


N/I means not indicated






B. Receptor Screening

Screening candidate compounds against a non-endogenous, constitutively activated version of the known GPCRs disclosed herein allows for the direct identification of candidate compounds which act at the cell surface of the receptor, without requiring use of the receptor's endogenous ligand. By determining areas within the body where the endogenous version of known GPCRs disclosed herein is expressed and/or over-expressed, it is possible to determine related disease/disorder states which are associated with the expression and/or over-expression of the receptor; such an approach is disclosed in this patent document.
Table C below lists the known GPCRs and tissues within the body are expressed and/or over-expressed. The listed references provide support for such tissue expression.









TABLE C





Known GPCRs
Location of Expression
Reference







5HT-1A
N/I
N/I


5HT-1B
Striatum
Jin, H. et al., 267(9) J Biol




Chem 5735 (1992)


5HT-1D
Cerebral cortex
Weinshank, R. L. et al., 89(8)




Proc Natl Acad Sci USA




3630 (1992)


5HT-1E
N/I
N/I


5HT-1F
Brain
Adham, N. et al., 90 Proc




Natl Acad Sci USA 408




(1993)


5HT-2B
Various tissues, including
Kursar, J. D., 46(2_Mol



Brain
Pharmacol 227 (1994)


5HT-4A
Brain, Intestine and Atrium
Blondel, O. et al., 70 J




Nerochem 2252 (1998)


5HT-4B
Brain, Intestine and Atrium
Blondel, O. et al., 70 J




Nerochem 2252 (1998)


5HT-4C
Brain, Intestine and Atrium
Blondel, O. et al., 70 J




Nerochem 2252 (1998)


5HT-4D
Intestine
Blondel, O. et al., 70 J




Neurochem 2252 (1998)


5HT-4E
Brain
Claeysen, S. et al., 55(5) Mol




Pharmacol 910 (1999)


5HT-5A
Brain
Rees, S. et al., 355(3) FEBS




Lett 242 (1994)


5HT-6
Caudate Nucleus
Kohen, R. et al., 66(1) J




Neurochem 47 (1996)


5HT-7
Brain, Coronary artery
Bard, J. A. et al., 268(31) J




Biol Chem 23422 (1993)


AVPR1A
N/I
N/I


AVPR1B
Pituitary
Sugimoto, T. et al., 269(43) J.




Biol. Chem 27088 (1994)


AVPR2
Lung, Kidney
Fay, M. J., et al., 17(3)




Peptides 477 (1996)


BBR3
Testis, Lung carcinoma
Fathi, Z. et al., 268(8) J. Biol.




Chem. 5979 (1993)


BDKR1
N/I
N/I


BDKR2
N/I
N/I


C3a
Lung, Spleen, Ovary,
Ames, R. et al., 271(34) J.



Placenta, Small Intestine and
Biol. Chem 20231 (1996)



Brain


C5a
N/I
N/I


CB1
Brain
Gerard, C. M. et al., 279




Biochem J. 129 (1991)


CB2
Spleen, Macrophage
Munro, S. et al., 365(6441)




Nature 61 (1993)


CCR2b
N/I
N/I


CCR3
Endometrium
Zhang, J. et al., 62(2) Biol




Reprod 404 (2000)


CCR5
Thymus, Spleen
Raport, C. J. et al., 271(29) J




Biol Chem 17161 (1996)


CCR8
Thymus, Spleen and Lymph
Napolitano M. et al., Forum



nodes
(Geneva) 1999 Oct–Dec;




9(4): 315–24


CCR9
Thymus
Zaballos, A. et al., 162(10) J.




Immunol 5671 (1999)


CRFR1
Brain, Pituitary
Perrin, M. H. et al., 133(6)




Endocrinology 3058 (1993)


CXCR4
Colonic epithelial cells
Jordan, N. J. et al., 104(8) J




Clin Invest 1061 (1999)


Dopamine D1
Caudate, Nucleus accumbens
Dearry, A. et al., 347 Nature



and Olfactory tubercle
72 (1990)


Dopamine D2
Retina
Dearry, A. et al., 11(5) Cell




Mol. Neurobiol. 437 (1991)


Dopamine D3
Brain
Schmauss, C. et al., 90(19)




Proc Natl Acad Sci USA




8942 (1993)


Dopamine D5
Brain
Sunahara, R. K. et al.,




350(6319) Nature 614 (1991)


ETA
Placenta, Uterus, Testis,
Adachi, M. et al., 180(3)



Adrenal gland
Biochem Biophys Res




Commun 1265 (1991)


ETB
N/I
N/I


FPR1
N/I
N/I


FPRL1
N/I
N/I


GALR1
Hypothalamic
Gundlach, A. L. et al., 863



paraventricular, Supraoptic
Ann NY Acad Sci 241



nuclei
(1998)


GALR2
Hypothalamus,
Fathi, Z. et al., 51 Brain Res



Hippocampus, Anterior
Mol Brain Res 49 (1997)



pituitary


GIP
N/I
N/I


mGluR1
Brain
Stephan, D. et al., 35(12)




Neuropharmacology 1649




(1996)


GPR5
Leukocyte cells
Shan, L. et al., 268(3)




Biochem Biophys Res




Commun 938 (2000)


GPR24 (also known as MCH
Fore-brain, Hypothalamus
Kolakowki LF Jr. et al.,


or SLC-1)

398(2–3) FEBS Lett 253




(1996)


GRPR
Lung carcinoma cells
Corjay, M. H. et al., 266 Jo




Biol Chem 18771 (1991)


M1
Heart, Pancreas and Neuronal
Peralta, E. G. et al., Embo J.



cell lines
6(13) 3923 (1987)


M2
Heart, Pancreas and Neuronal
Peralta, E. G. et al., Embo J.



cell lines
6(13) 3923 (1987)


M3
Heart, Pancreas and Neuronal
Peralta, E. G. et al., Embo J.



cell lines
6(13) 3923 (1987)


M4
Heart, Pancreas and Neuronal
Peralta, E. G. et al., Embo J.



cell lines
6(13) 3923 (1987)


M5
Brain
Bonner, T. I. et al., Neuron




1(5), 403 (1988)


MC3
Brain, Placenta, Gut
Gantz I. et al., 268(11) Jo




Biol Chem 8246 (1993)


NK1R
Spinal cord, Lung
Taked, Y. et al., 179(3)




Biochem Biophys Res




Commun 1232 (1991)


NK2R
N/I
N/I


NK3R
N/I
N/I


NMBR
Lung carcinoma cells
Corjay, M. H. et al., 266 Jo




Biol Chem 18771 (1991)


NPY5
Hypothalamus
Gerald, C. et al., 382 Nature




168 (1996)


NTSR1
Brain, Small intestine
Vita, N. et al., 17 (1–2) FEBS




Lett 139 (1993)


NTSR2
N/I
N/I


OPRD
Peripheral blood
Wick, M. J. et al., 64(1) J.



lymphocytes
Neuroimmunol 29(1996)


OPRL1
N/I
N/I


OPRK
Placenta, Brain
Manson, E. et al., 202(3)




Biochem Biophys Res




Commun 1431 (1994)


OPRM
N/I
N/I


OPRM1A
N/I
N/I


OX1R
Hypothalamus
Sakurai T. et al., 92 Cell 573




(1998)


OX2R
Hypothalamus
Sakurai T. et al., 92 Cell 573




(1998)


PACAP
Brain
Ogi, K. et al., 196(3)




Biochem Biophys Res




Commun 1511 (1993)


PAF
N/I
N/I


PGE EP1
Kidney
Watabe, A. et al., 268(27) J




Biol Chem 20175 (1993)


PGE EP2
Small Intestine
Bastien, L. et al., 269(16) J.




Biol. Chem 11873 (1994)


PGE EP4
N/I
N/I


PTHR1
Bone, Kidney
Schipani, E. et al., 132(5)




Endocrinology 2157 (1993)


PTHR2
Brain, Pancreas
Usdin, T. B. et al., 270(26) J.




Biol Chem 15455 (1995)


SCTR
Pancreas, Intestine
Chow, B. K., 212(1) Biochem




Biophys Res Commun 204




(1995)


SST1
Jejunum, Stomach
Yamada Y. et al., 89 Proc




Natl Acad Sci USA 251




(1992)


SST2
Cerebrum, Kidney
Yamada Y. et al., 89 Proc




Natl Acad Sci USA 251




(1992)


SST3
Brain, Pancreatic islet
Yamada Y. et al., 6 Mol




Endocrinol 2136 (1992)


SST4
Fetal, Adult Brain, Lung
Rohser L. et al., 90(9) Pro




Natl Acad Sci USA 4146




(1993)


SST5
Pituitary
Panetta R. et al., 45(3) mol




Pharmacol 417 (1994)


TSHR
Retro-orbital tissues,
Feliciello A. et al 342 Lancet



Exophthalmos
337 (1993)


VIPR
Lung
Sreedharan, S. P. et al., 193(2)




Biochem Biophys Res




Commun 546 (1993)


VIPR2
Skeletal muscle
Adamou, J. E. et al., 209(2)




Biochem Biophys Res




Commun 385 (1995)





Note:


N/I means not indicated






Creation of a non-endogenous version of a known GPCR that may evidence constitutive activity is most preferably based upon the distance from a proline residue located within TM6 of the GPCR; this technique is disclosed in co-pending and commonly assigned patent document U.S. Ser. No. 09/170,496, incorporated herein by reference. This technique is not predicated upon traditional sequence “alignment” but rather a specified distance from the aforementioned TM6 proline residue. By mutating the amino acid residue located 16 amino acid residues from this residue (presumably located in the IC3 region of the receptor) to, most preferably, a lysine residue, such activation may be obtained. Other amino acid residues may be used for the mutation, but lysine is most preferred.
D. Disease/Disorder Identification and/or Selection
As will be set forth in greater detail below, most preferably inverse agonists, partial agonists and agonists in the form of small molecule chemical compounds to the non-endogenous, constitutively activated GPCR can be identified by the methodologies of this invention. Such compounds are ideal candidates as lead modulators in drug discovery programs for treating diseases or disorders associated with a particular receptor. The ability to directly identify such compounds to the GPCR, in the absence of use of the receptor's endogenous ligand, allows for the development of pharmaceutical compositions.
Preferably, in situations where it is unclear what disease or disorder may be associated with a receptor; the DNA sequence of the known GPCR is used to make a probe for (a) dot-blot analysis against tissue-mRNA, and/or (b) RT-PCR identification of the expression of the receptor in tissue samples. The presence of a receptor in a tissue source, or a diseased tissue, or the presence of the receptor at elevated concentrations in diseased tissue compared to a normal tissue, can be preferably utilized to identify a correlation with a treatment regimen, including but not limited to, a disease associated with that disease. Receptors can equally well be localized to regions of organs by this technique. Based on the known functions of the specific tissues to which the receptor is localized, the putative functional role of the receptor can be deduced.
E. Screening of Candidate Compounds
1. Generic GPCR Screening Assay Techniques
When a G protein receptor becomes constitutively active, it binds to a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP. A non-hydrolyzable analog of GTP, [35S]GTPγS, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [35S]GTPγS can be used to monitor G protein coupling to membranes in the absence and presence of ligand. An example of this monitoring, among other examples well-known and available to those in the art, was reported by Traynor and Nahorski in 1995. The preferred use of this assay system is for initial screening of candidate compounds because the system is generically applicable to all G protein-coupled receptors regardless of the particular G protein that interacts with the intracellular domain of the receptor.
2. Specific GPCR Screening Assay Techniques
Once candidate compounds are identified using the “generic” G protein-coupled receptor assay (i.e., an assay to select compounds that are agonists, partial agonists, or inverse agonists), further screening to confirm that the compounds have interacted at the receptor site is preferred. For example, a compound identified by the “generic” assay may not bind to the receptor, but may instead merely “uncouple” the G protein from the intracellular domain.
a. Gs, Gz and Gi.
Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on the other hand, inhibit this enzyme. Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, constitutively activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP. On the other hand, constitutively activated GPCRs that couple Gi (or Gz, Go) protein are associated with decreased cellular levels of cAMP. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can be utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP). A variety of approaches known in the art for measuring cAMP can be utilized; a most preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format. Another type of assay that can be utilized is a second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes. Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene. Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., β-galactosidase or luciferase. Thus, a constitutively activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of the reporter protein. The reporter protein such as β-galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).
b. Go and Gq.
Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP2, releasing two intracellular messengers: diacycloglycerol (DAG) and inistol 1,4,5-triphoisphate (IP3). Increased accumulation of IP3 is associated with activation of Gq- and Go-associated receptors. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Assays that detect IP3 accumulation can be utilized to determine if an candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP3). Gq-associated receptors can also be examined using an AP1 reporter assay in that Gq-dependent phospholipase C causes activation of genes containing AP1 elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression. Commercially available assays for such detection are available.
3. Ligand-Based Confirmation Assays
The candidate compounds directly identified using the techniques (or equivalent techniques) above are then, most preferably, verified using a ligand-based verification assay, such as the one set forth in the protocol of Example 8. The importance here is that the candidate compound be directly identified; subsequent confirmation, if any, using the endogenous ligand, is merely to confirm that the directly identified candidate compound has targeted the receptor.
For example, sumatriptan is a well-known agonist of the 5-HT1B and 5-HT1D receptors, while naltrindole is a well-known antagonist to the OPM1D receptor. Accordingly, an agonist (sumatriptan) and/or antagonist (naltrindole) competitive binding assay(s) can be used to confirm that those candidate compounds directly identified using a ligand independent screening technique comprising non-endogenous, constitutively activated 5-HT1B or 5-HT1D, and non-endogenous constitutively activated OPM1D, respectfully, may be used for confirmatory purposes. Those skilled in the art are credited with the ability to select techniques for ligand-based confirmation assays.
4. GPCR Fusion Protein
The use of a non-endogenous, constitutively activated GPCR, for use in screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists, provides an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, in order to differentiate between, e.g., the non-endogenous receptor in the presence of a candidate compound and the non-endogenous receptor in the absence of that compound, with an aim of such a differentiation to allow for an understanding as to whether such compound may be an inverse agonist, agonist, partial agonist or has no affect on such a receptor, it is preferred that an approach be utilized that can enhance such differentiation. A preferred approach is the use of a GPCR Fusion Protein.
Generally, once it is determined that a non-endogenous GPCR has been constitutively activated using the assay techniques set forth above (as well as others), it is possible to determine the predominant G protein that couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed. Because it is most preferred that screening take place by use of a mammalian expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the non-endogenous, constitutively activated GPCR will continuously signal. In this regard, it is preferred that this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.
The GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the non-endogenous GPCR. The GPCR Fusion Protein is preferred for screening with a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening techniques. This is important in facilitating a significant “signal to noise” ratio; such a significant ratio is preferred for the screening of candidate compounds as disclosed herein.
The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator. The criteria of importance for such a GPCR Fusion Protein construct is that the endogenous GPCR sequence and the G protein sequence both be in-frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence) and that the “stop” codon of the GPCR must be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed. The GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). Use of a spacer is preferred (based upon convenience) in that some restriction sites that are not used will, effectively, upon expression, become a spacer. Most preferably, the G protein that couples to the non-endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is preferred that a construct comprising the sequence of the G protein (i.e., a universal G protein construct) be available for insertion of an endogenous GPCR sequence therein; this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.
F. Co-Transfection of a Target Gi Coupled GPCR with a Signal-Enhancer Gs Coupled GPCR (cAMP Based Assays)
A Gi coupled receptor is known to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging. An effective technique in measuring the decrease in production of cAMP as an indication of constitutive activation of a receptor that predominantly couples Gi upon activation can be accomplished by co-transfecting a signal enhancer, e.g., a non-endogenous, constitutively activated receptor that predominantly couples with Gs upon activation (e.g., TSHR-A623I, disclosed below), with the Gi linked GPCR (such a technique is exemplified herein with the Gi coupled receptor, GPR24). As is apparent, constitutive activation of a Gs coupled receptor can be determined based upon an increase in production of cAMP. Constitutive activation of a Gi coupled receptor leads to a decrease in production cAMP. Thus, the co-transfection approach is intended to advantageously exploit these “opposite” affects. For example, co-transfection of a non-endogenous, constitutively activated Gs coupled receptor (the “signal enhancer”) with the endogenous Gi coupled receptor (the “target receptor”) provides a baseline cAMP signal (i.e., although the Gi coupled receptor will decrease cAMP levels, this “decrease” will be relative to the substantial increase in cAMP levels established by constitutively activated Gs coupled signal enhancer). By then co-transfecting the signal enhancer with a constitutively activated version of the target receptor, cAMP would be expected to further decrease (relative to base line) due to the increased functional activity of the Gi target (i.e., which decreases cAMP).
Screening of candidate compounds using a cAMP based assay can then be accomplished, with two provisos: first, relative to the Gi coupled target receptor, “opposite” effects will result, i.e., an inverse agonist of the Gi coupled target receptor will increase the measured cAMP signal, while an agonist of the Gi coupled target receptor will decrease this signal; second, as would be apparent, candidate compounds that are directly identified using this approach should be assessed independently to ensure that these do not target the signal enhancing receptor (this can be done prior to or after screening against the co-transfected receptors).
G. Medicinal Chemistry
Generally, but not always, direct identification of candidate compounds is preferably conducted in conjunction with compounds generated via combinatorial chemistry techniques, whereby thousands of compounds are randomly prepared for such analysis. Generally, the results of such screening will be compounds having unique core structures; thereafter, these compounds are preferably subjected to additional chemical modification around a preferred core structure(s) to further enhance the medicinal properties thereof. Such techniques are known to those in the art and will not be addressed in detail in this patent document.
H. Pharmaceutical Compositions
Candidate compounds selected for further development can be formulated into pharmaceutical compositions using techniques well known to those in the art. Suitable pharmaceutically-acceptable carriers are available to those in the art; for example, see Remington's Pharmaceutical Sciences, 16th Edition, 1980, Mack Publishing Co., (Oslo et al., eds.).
I. Other Utility
Although a preferred use of the non-endogenous version of the known GPCRs disclosed herein may be for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as pharmaceutical agents), these versions of known GPCRs can also be utilized in research settings. For example, in vitro and in vivo systems incorporating GPCRs can be utilized to further elucidate and better understand the roles these receptors play in the human condition, both normal and diseased, as well as understanding the role of constitutive activation as it applies to understanding the signaling cascade. The value in non-endogenous known GPCRs is that their utility as a research tool is enhanced in that, because of their unique features, non-endogenous known GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand therefor is identified. Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document.
EXAMPLES
The following examples are presented for purposes of elucidation, and not limitation, of the present invention. While specific nucleic acid and amino acid sequences are disclosed herein, those of ordinary skill in the art are credited with the ability to make minor modifications to these sequences while achieving the same or substantially similar results as reported below. The traditional approach to application or understanding of sequence cassettes from one sequence to another (e.g. from rat receptor to human receptor or from human receptor A to human receptor B) is generally predicated upon sequence alignment techniques whereby the sequences are aligned in an effort to determine areas of commonality. The mutational approaches disclosed herein do not rely upon a sequence alignment approach but are instead based upon an algorithmic approach and a positional distance from a conserved proline residue located within the TM6 region of GPCRs. Once this approach is secured, those in the art are credited with the ability to make minor modifications thereto to achieve substantially the same results (i.e., constitutive activation) disclosed herein. Such modified approaches are considered within the purview of this disclosure
Example 1
Preparation of Endogenous known GPCRs
A. Expression By Standard PCR
PCR was performed using a specific cDNA as template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides.
The resulting PCR fragment was digested with the respective restriction sites and cloned into a pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified. See Table D below:











TABLE D








5′ Primer
3′ Primer


Receptor

Cycle Conditions
(SEQ. ID. NO.) and
(SEQ. ID. NO.) and


Identifier
Template
Min (′), Sec (′′)
Restriction site
Restriction site







5HT-1A
Genomic DNA
94° for 1′
CGGAAGCTTAGC
CCGGAATTCCTG




60° C. for 1′
CATGGATGTGCT
GCGGCAGAAGTT




72° for 1′30′′
CAGCCCTGGTCA
ACACTTAATG (2);





(1); HindIII
EcoRI


 


5HT-1B
Genomic DNA
94° for 1′
TCCAAGCTTGGG
GGCGAATTCACTT




60° C. for 1′
GCGAGGAGAGCC
GTGCACTTAAAA




72° for 1′30′′
ATGGAGGA (3);
CGTATCAGTT (4);





HindIII
EcoRI


 


5HT-1D
Genomic DNA
94° for 1′
ATCTACCATGTC
ATAGAATTCGGA




60° C. for 1′
CCCACTGAACCA
GGCCTTCCGGAA




72° for 1′30′′
GTCAGC (5)
AGGGACAA (6);






EcoRI


 


5HT-1E
Genomic DNA
94° for 1′
CCACAGTGTCGA
CAGTATGCTCTCG




60° C. for 1′
CTGAAACAAGGG
GCATCTAATGAG




72° for 2′10′′
AAACATGAAC
(8)





(7); SalI


 


5HT-1F
Genomic DNA
94° for 1′
ATCACCATGGAT
TTAGGATCCACAT




60° C. for 1′
TTCTTAAATTCAT
CGACATCGCACA




72° for 2′10′′
CTGATC (9)
AGCTTTTG (10);






BamHI


 


5HT-2B
Uterus cDNA
94° for 1′
GAAAAGCTTGCC
GTTGGATCCTACA




60° C. for 1′
ATGGCTCTCTCTT
TAACTAACTTGCT




72° for 1′30′′
ACAGAGTGTCTG
CTTCAGTTT (12);





(11); HindIII
BamHI


 


5HT-4A
Brain cDNA
94° for 1′
ATCACCATGGAC
CCTGAATTCGAA




60° C. for 1′
AAACTTGATGCT
GCATGATTCCAG




72° for 1′30′′
AATGTGAG (13)
GGATTCTGG (14);






EcoRI


 


5HT-4B
Brain cDNA
94° for 1′
ATCACCATGGAC
AGGGAATTCAGT




60° C. for 1′
AAACTTGATGCT
GTCACTGGGCTG




72° for 1′30′′
AATGTGAG (15)
AGCAGCCAC (16);






EcoRI


 


5HT-4C
Brain cDNA
94° for 1′
ATCACCATGGAC
TTGGAATTCGGAT




60° C. for 1′
AAACTTGATGCT
GGTTTGGTCAATC




72° for 1′30′′
AATGTGAG (17)
TTCTCTTC (18);






EcoRI


 


5HT-4D
5HT-4E DNA
94° for 1′
ATCACCATGGAC
AGGGAATTCAAA




60° C. for 1′
AAACTTGATGCT
TCTTAGTACATGT




72° for 2′′
AATGTGAG (19)
GTGGATCCATTA






AT (20); EcoRI


 


5HT-4E
Brain cDNA
94° for 1′
ATCACCATGGAC
TCAGAATTCGAC




60° C. for 1′
AAACTTGATGCT
AGGAACTGGTCT




72° for 1′15′′
AATGTGAG (21)
ATTGCAGAA (22);






EcoRI


 


5HT-5A
Brain cDNA
94° for 1′
CCTAAGCTTGCC
TCTGAATTCGTGT




60° C. for 1′
ATGGATTTACCA
TGCCTAGAAAAG




72° for 2′10′′
GTGAACCTAACC
AAGTTCTTGA





TCC (23); HindIII
(24); EcoRI


 


5HT-6
See alternative
See alternative
See alternative
See alternative



approach below
approach below
approach below
approach below


 


5HT-7
Brain cDNA
94° C. for 1′
AGCGGAATTCGG
TTTCGGATCCATT




72° C. for 2′′
CGGCGCGATGAT
GTTCTGCTTTCAA





GGACGTT (25);
TCAT (26); BamHI





EcoRI


 


AVPR1A
See alternative
See alternative
See alternative
See alternative



approach below
approach below
approach below
approach below


 


AVPR1A
See alternative
See alternative
See alternative
See alternative


variant
approach below
approach below
approach below
approach below


 


AVPR1B
See alternative
See alternative
See alternative
See alternative



approach below
approach below
approach below
approach below


 


AVPR2
IMAGE 301449
pfu PCR
CAGGAATTCAGA
AGCGGATCCCGA




94° for 1′
ACACCTGCCCCA
TGAAGTGTCCTTG




63° C. for 1′
GCCCCAC (27);
GCCAGGGA (28);




72° for 2′′
EcoRI
BamHI


 


BBR3
Uterus cDNA
94° C. for 1′
ACAGAATTCAGA
CATGGATCCTTGA




56° C. for 1′
AGAAATGGCTCA
AAAGCTAGAAAC




72° C. for 1′20′′
AAGGCA (29);
TGTCC (30); BamHI





EcoRI


 


BDKR1
IMAGE 1472696
pfu PCR
TGTAAGCTTCAG
GCTGGATCCATTC




94° for 1′
GTCACTGTGCAT
CGCCAGAAAAGT




65° C. for 1′
GGCATCATC (31);
TGGAAGATTTC




72° for 2′′
HindIII
(32); BamHI


 


BDKR2
IMAGE 1682455
pfu PCR
ACTAAGCTTCCA
GTTGAATTCCTGT




94° for 1′
AATGTTCTCTCCC
CTGCTCCCTGCCC




65° C. for 1′
TGGAAGATA (33)
AGTCCTG (34);




72° for 2′′
HindIII
EcoRI


 


C3a
Genomic DNA
94° for 1′
CAGAAGCTTAGC
ACAGGATCCCAC




65° C. for 1′
AATGGCGTCTTT
AGTTGTACTATTT




72° for 1′30′′
CTCTGCTG (35);
CTTTCTGAAATG





HindIII
(36); BamHI


 


C5a
Thymus
94° for 1′
GGGAAGCTTAGG
TGTGAATTCCACT




65° C. for 1′
AGACCAGAACAT
GCCTGGGTCTTCT




72° for 1′10′′
GAACTCCTTC
GGGCCAT (38);





(37); HindIII
EcoRI


 


CB1
EST 01536
pfu PCR
GGGAAGCTTTCT
TCAGAATTCCAG




94° for 1′
CAGTCATTTTGA
AGCCTCGGCAGA




65° C. for 1′
GCTCAGCC (39);
CGTGTCTGT (40);




72° for 2′30′′
HindIII
EcoRI


 


CB2
IMAGE 1301708
pfu PCR
CAAAAGCTTCTA
GCCGAATTCGCA




94° for 1′
GACAAGCTCAGT
ATCAGAGAGGTC




60° C. for 1′
GGAATCTGA (41);
TAGATCTCTG




72° for 2′′
HindIII
(42); EcoRI


 


CCR2b
Genomic DNA
94° for 1′
GACAAGCTTCCC
CTCGGATCCTAA




60° C. for 1′
CAGTACATCCAC
ACCAGCCGAGAC




72° for 1′10′′
AACATGC (43);
TTCCTGCTC (44);





HindIII
BamHI


 


CCR3
Genomic DNA
94° for 1′
ATCGCCATGACA
TCTGAATTCAAAC




60° C. for 1′
ACCTCACTAGAT
ACAATAGAGAGT




72° for 1′10′′
ACAGTTGAG (45)
TCCGGCTC (46);






EcoRI


 


CCR5
Genomic DNA
94° for 1′
GCAAAGCTTGGA
TCCGGATCCCAA




62° C. for 1′
ACAAGATGGATT
GCCCACAGATAT




72° for 1′10′′
ATCAAGTGTC
TTCCTGCTC (48);





(47); HindIII
BamHI


 


CCR8
Genomic DNA
94° for 1′
TGAAAGCTTCCC
TGAGAATTCCAA




60° C. for 1′
GCTGCCTTGATG
AATGTAGTCTAC




72° for 1′10′′
GATTATAC (49);
GCTGGAGGAA





HindIII
(50); EcoRI


 


CCR9
Genomic DNA
94° for 1′
ATCACCATGACA
GACGAATTCGAG




60° C. for 1′
CCCACAGACTTTC
GGAGAGTGCTCC




72° for 1′10′′
ACAAGCCCTATT
TGAGGTTGT (52);





CCTAACATGGCT
EcoRI





GATGACTATGG





(51)


 


CRFR1
Pituitary cDNA
94° for 1′
ATCACCATGGGA
CGGGAATTCGAC




65° C. for 1′
GGGCACCCGCAG
TGCTGTGGACTGC




72° for 1′20′′
CTCCGT (53)
TTGATGCT (54);






EcoRI


 


CXCR4
Genomic DNA
94° for 1′
ATCACCATGGAG
TCTGAATTCGCTG




65° C.
GGGATCAGTATA
GAGTGAAAACTT




72° for 1′
TACACTTCAGAT
GAAGACTCAG





AACTACACCGAG
(56); EcoRI





GAAATG (55)


 


Dopamine
See alternative
See alternative
See alternative
See alternative


D1
approach below
approach below
approach below
approach below


 


Dopamine
See alternative
See alternative
See alternative
See alternative


D2
approach below
approach below
approach below
approach below


 


Dopamine
Brain cDNA
94° C. for 1′
AAGAAGCTTGGC
GGCTCTAGAAAT


D3

62° for 1′20′′
ATCACGCACCTC
GGGTACAAAGAG




72° C. for 1′20′′
CTCTGG (57);
TGTT (58);





HindIII
XbaI


 


Dopamine
Genomic DNA
See alternative
See alternative
See alternative


D5

approach below
approach below
approach below


 


ETA
See alternative
See alternative
See alternative
See alternative



approach below
approach below
approach below
approach below


 


ETB
IMAGE 1086987
pfu PCR
CGGAAGCTTCTG
CTTGGATCCAGAT




94° for 1′
GAGCAGGTAGCA
GAGCTGTATTTAT




60° C. for 1′
GCATG (59);
TACTGGAACG




72° for 2′20′′
HindIII
(60); BamHI


 


FPR1
IMAGE 2153284
94° C. for 1′
ATCACCATGGAG
CCCGAATTCCTTT




63° for 1′
ACAAATTCCTCT
GCCTGTAACTCCA




72° C. for 2′30′′
CTCCCC (61)
CCTCTGC (62);






EcoRI


 


FPRL1
Genomic DNA
94° C. for 1′
GCAAAGCTTGCT
CCAGAATTCCATT




65° for 1′
GCTGGCAAGATG
GCCTGTAACTCA




72° C. for 1′10′′
GAAACCAAC (63);
GTCTCTGC (64);





HindIII
EcoRI


 


GALR1
Stomach cDNA
94° C. for 1′
CCGGAATTCGCC
GCAGGATCCTTAT




60° for 1′20′′
GGGACAGCCCCG
CACACATGAGTA




72° C. for 1′20′′
CGGGCC (65);
CAATTGGT (66);





EcoRI
BamHI


 


GALR2
Hippocampus
94° C. for 1′
GGCGAATTCGGG
GTGGGATCCCAG



cDNA
62° for 1′20′′
GTCAGCGGCACC
CGCGCCCGCTAA




72° C. for 1′20′′
ATGAACG (67);
GTGCT (68); BamHI





EcoRI


 


GIP
Brain cDNA
94° for 1′
CAGAAGCTTCGC
CGCGAATTCGCA




65° C. for 1′
CGCCCTCACGAT
GTAACTTTCCAAC




72° for 1′30′′
GACTAC (69);
TCCCGGCT (70);





HindIII
EcoRI


 


mGluR1
See alternative
See alternative
See alternative
See alternative



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GPR5
Genomic DNA
94° C. for 1′
TATGAATTCAGA
TCCGGATCCACCT




64° for 1′
TGCTCTAAACGT
GCACCTGCGCCT




72° C. for 1′30′′
CCCTGC (71);
GCACC (72); BamHI





EcoRI


 


GPR24 (also
See alternative
See alternative
See alternative
See alternative


known as
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MCH or


SLC-1)


 


GRPR
Stomach cDNA
94° C. for 1′
AGGAAGCTTTTA
CCGGAATTCAAG




56° for 1′20′′
GGTGGGAAAAA
GGGCAAAATCAA




65° C. for 1′20′′
AAATCTA (73);
GGGTCAA (74);





HindIII
EcoRI


 


M1
Genomic DNA
94° C. for 1′
GCCAAGCTTAGC
GGAGAATTCGCA




60° for 1′
CACCATGAACAC
TTGGCGGGAGGG




72° C. for 1′50′′
TTCAGCCC (75);
AGTGCGGTG (76);





HindIII
EcoRI


 


M2
Genomic DNA
94° C. for 1′
ATCACCATGAAT
GATGAATTCCCTT




60° for 1′
AACTCAACAAAC
GTAGCGCCTATGT




72° C. for 1′50′′
TCCTCTAAC (77)
TCTTATA (78);






EcoRI


 


M3
Genomic DNA
94° C. for 1′
ATCACCATGACC
CTCGAAATTCCA




60° for 1′
TTGCACAATAAC
AGGCCTGCTCGG




72° C. for 1′50′′
AGTACAAC (79)
GTGCGCGCT (80);






EcoRI


 


M4
Genomic DNA
94° C. for 1′
ATCACCATGGCC
GCCGAATTCCCTG




60° for 1′
AACTTCACACCT
GCAGTGCCGATG




72° C. for 1′50′′
GTCAA (81)
TTCCGATA (82);






EcoRI


 


M5
Genomic DNA
94° C. for 1′
ATCACCATGGAA
GACGGATCCGGG




60° for 1′
GGGGATTCTTAC
TAGCTTGCTGTTC




72° C. for 1′50′′
CACAAT (83)
CCCTGCCA (84);






BamHI


 


MC3
Genomic DNA
94° C. for 1′
CAGGAATTCTGA
AATGGATCCTATC




54° for 1′30′′
CAGCAATGAATG
CCAAGTTCATGCC




72° C. for 1′20′′
CTTCGT (85);
GTTGCAG (86);





EcoRI
BamHI


 


NK1R
Brain cDNA
94° C. for 1′
AGTAAGCTTTAC
TGTGAATTCGGA




65° for 1′
GCCTAGCTTCGA
GAGCACATTGGA




72° C. for 1′50′′
AATGGAT (87);
GGAGAAGCT (88);





HindIII
EcoRI


 


NK2R
Uterus cDNA
94° C. for 1′
TCCAAGCTTAGA
AACGAATTCAAT




65° for 1′
AGCAGCCATGGG
TTCAACATGAGTT




72° C. for 1′50′′
GACCTGTGACA
TTGGTGGGGG





(89); HindIII
(90); EcoRI


 


NK3R
Brain cDNA
94° C. for 1′
ATCTGCAGACCG
ATGGGATCCAGA




65° for 1′20′′
GTGGCGATGGCC
ATATTCATCCACA




72° C. for 1′20′′
ACT (91)
GAGGTATAGG





(92); BamHI


 


NMBR
Brain cDNA
94° C. for 1′
TGAGAATTCCAG
GTTGGATCCAGG




65° for 1′20′′
CGGACTCTGCTG
TAGTGAGTTGAA




72° C. for 1′20′′
GAAAGGA (93);
TGGCCA (94);





EcoRI
BamHI


 


NPY5
Genomic DNA
94° C. for 1′
GGAAAGCTTCAA
GGAGGATCCAGT




54° for 1′30′′
GAAAGACTATAA
GAGAATTATTAC




72° C. for 1′20′′
TATGGAT (95);
ATATGAAG (96);





HindIII
BamHI


 


NTSR1
See alternative
See alternative
See alternative
See alternative



approach below
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NTSR2
See alternative
See alternative
See alternative
See alternative



approach below
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approach below


 


OPRD
Brain cDNA
94° C. for 1′
CGGAAGCTTGCA
GCCGAATTCGGC




65° for 1′
GCCATGGAACCG
GGCAGCGCCACC




72° C. for 1′15
GCCCCCTCC (97);
GCCGGGACC (98);





HindIII
EcoRI


 


OPRL1
Brain cDNA
94° C. for 1′
AGTAAGCTTGCA
GCCGAATTCTGC




65° for 1′
GGGCAGTGGCAT
GGGGCGCGGTAC




72° C. for 1′15′′
GGAGCCC (99);
CGTCTCAGA (100);





HindIII
EcoRI


 


OPRK
Brain cDNA
94° C. for 1′
TTTAAGCTTGCA
CTACTGGTTTATT




65° for 1′
GCACTCACCATG
CATCCCATCGATG




72° C. for 1′15′′
GAATCCCCGAT
TC (102);





(101); HindIII


 


OPRM
See alternative
See alternative
See alternative
See alternative



approach below
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OPRM1A
See alternative
See alternative
See alternative
See alternative



approach below
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OX1R
See alternative
See alternative
See alternative
See alternative



approach below
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OX2R
Brain cDNA
94° C. for 1′
ACCAAGCTTGAG
CAGGGATCCTTGT




65° for 1′
CCCGTGATGTCC
CATATGAATAAA




72° C. for 1′20′′
GGCACC (103);
TATT (104); BamHI





HindIII


 


PACAP
Fetal Brain cDNA
94° C. for 1′
AGTAAGCTTGGC
CATGAATTCGGT




65° for 1′
CAAGAAGTGTCA
GGCCAGATTGTC




72° C. for 1′30′′
TGGCTGGTG
AGCAGGGAG





(105); HindIII
(106); EcoRI


 


PAF
Genomic DNA
94° C. for 1′
CTGAAGCTTCCA
CAGGAATTCATTT




63° for 1′
GCCCACAGCAAT
TTGAGGGAATTG




72° C. for 2′30′′
GGAGCCA (107);
CCAGGGATCTG





HindIII
(108); EcoRI


 


PGE EP1
cDNA clone
pfu PCR
ATCGCCATGAGC
TTGGAATTCGAA




94° C. for 1′
CCTTGCGGGCCC
GTGGCTGAGGCC




63° for 1′
CTCAA(109)
GCTGTGCCG (110);




72° C. for 2′30′′

EcoRI


 


PGE EP2
Thymus cDNA
94° C. for 1′
GCAAAGCTTTTC
CTGGAATTCAAG




63° for 1′
CAGGCACCCCAC
GTCAGCCTGTTTA




72° C. for 2′30′′
CATGGGC (111);
CTGGCATC (112);





HindIII
EcoRI


 


PGE EP4
cDNA clone
pfu PCR
ATCATCATGTCC
TGCGAATTCTATA




94° C. for 1′
ACTCCCGGGGTC
CATTTTTCTGATA




60° for 1′
AAT (113)
AGTTCAGTGTT




72° C. for 2′30′′

(114); EcoRI


 


PTHR1
See alternative
See alternative
See alternative
See alternative



approach below
approach below
approach below
approach below


 


PTHR2
Brain cDNA
94° C. for 1′
CTGAAGCTTCCT
CGAGAACATCCT




65° for 1′
ACAGCCGTTCCG
CAGTTTCTCCTTG




72° C. for 1′50′′
GGCATG (115);
G (116)





HindIII


 


SCTR
Small Intestine
94° C. for 1′
GGGAAGCTTGCG
AGCGAATTCGAT




65° for 1′
GGCACCATGCGT
GATGCTGGTCCTG




72° C. for 1′45′′
CCCCACCT (117);
CAGGTGCC (118);





HindIII
EcoRI


 


SST1
Genomic DNA
94° C. for 1′
GCCGAATTCAGC
CAGGGATCCTGC




65° for 1′20′′
TGGGATGTTCCC
GTGGCCCGGGCT




72° C. for 1′20′′
CAATGGC (119);
CAGAGCG (120);





EcoRI
BamHI


 


SST2
See alternative
See alternative
See alternative
See alternative



approach below
approach below
approach below
approach below


 


SST3
Genomic DNA
94° C. for 1′
ACGGAATTCCCC
TGGGATCCCCAG




65° for 1′20′′
TCAGCCATGGAC
GCCCCTACAGGT




72° C. for 1′20′′
ATGCTTC (121);
AGCTG (122);





EcoRI
BamHI


 


SST4
Genomic DNA
94° C. for 1′
GCCGAATTCAGC
GAGGGATCCACG




65° for 1′20′′
TGCCCTGCGCCG
CAGGGTGGGTAG




72° C. for 1′20′′
GCACCCC (123);
GGGAAGG (124);





EcoRI
BamHI


 


SST5
Genomic DNA
94° C. for 1′
TCTAAGCTTGCA
CCTGAATTCCTGG




65° for 1′20′′
GAGCCTGACGCA
GGGTGACACGGG




72° C. for 1′20′′
CCCCAG (125);
GCCGCC (126);





HindIII
EcoRI


 


TSHR
Genomic DNA
94° C. for 1′
GGCGAATTCGGA
GTAGGATCCCCT




65° for 1′
GGATGGAGAAAT
ACCATTGTGAGT




72° C. for 2′30′′
AGCCCC (127);
AGTGTA (128);





EcoRI
BamHI


 


VIPR1
Lung cDNA
94° C. for 1′
CCGAAGCTTCAG
TGGGAATTCGAC




65° for 1′
GGCAGACCATGC
CAGGGAGACTTC




72° C. for 1′30′′
GCCCGCCA (129);
GGCTTTGGAA





HindIII
(130);EcoRI


 


VIPR2
Brain cDNA
94° C.for 1′
GCTAAGCTTGCC
GTGGAATTCGAT




65° for 1′
ATGCGGACGCTG
GACCGAGGTCTC




72° C. for 1′30′′
CTGCCTCCCGCG
CGTTTGCAG (132);





(131); HindIII
EcoRI









B. Expression by Alternative Approaches
1. AVPR1A
The endogenous human AVPR1A was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 64° C. for 1 min and 72° C. for 1 min and 30 sec. The 5′ PCR fragment was obtained utilizing genomic DNA, as a template, and the following primer set:







(SEQ. ID. NO.:133)









5′-ATCACCATGCGTCTCTCCGCCGGTCCCGA-3′



 



and



 







(SEQ. ID. NO.:134)









5′-TTGTTCACCTCGATCATGGAGAAGA-3′-.






The 3′ PCR fragment was obtained by pfu polymerase (Stratagene) using IMAGE 1055179, as a template, and the following primer set:








(SEQ. ID. NO.:135)









5′-CGCAGTACTTCGTCTTCTCCATGA-3′



 



and



 







(SEQ. ID. NO.:136)









5′-CAAGAATTCAGTTGAAACAGGAATGAATTTGATGG-3′.






The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 60° C. and 72° C. for 1 min 30 sec. The 5′ and 3′ PCR fragments were then used as co-templates to obtain the full length cDNA using the pfu polymerase and SEQ.ID.NO.:133 and SEQ.ID.NO.:136 as primers. The cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. and 72° C. for 2 min 10 sec.

The resulting PCR fragment was digested with EcoRI restriction site and cloned into an EcoRI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
2. AVPR1A Variant
The endogenous human AVPR1A variant was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 64° C. for 1 min and 72° C. for 1 min and 30 sec. The 5′ PCR fragment was obtained utilizing genomic DNA, as a template, and: SEQ.ID.NO.:133 and SEQ.ID.NO.:134 as primers.
The 3′ PCR fragment was obtained by pfu polymerase (Stratagene) using IMAGE 1542469, as a template, and SEQ.ID.NO.:136 and







(SEQ. ID. NO.:137)









5′-ACAGAATTCTCCAGTTCTCATTTTCTTATCCGTAC-3′.






The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min 30 sec. The 5′ and 3′ PCR fragments were then used as co-templates to obtain the full length cDNA using the pfu polymerase (Stratagene) and SEQ.ID.NO.:133 and SEQ.ID.NO.:136 as primers. The cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 2 min 10 sec.

The resulting PCR fragment was digested with EcoRI restriction site and cloned into an EcoRI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
3. AVPR1B
The endogenous human AVPR1B was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. Both rounds of PCR had the following cycle condition: 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 2 sec. The first round of PCR utilized a pituitary DNA, as a template, and a 5′ PCR primer that contained a HindIII site with the following sequence:







(SEQ. ID. NO.:138)









5′-GCAAAGCTTGCTCATGGATTCTGGGCCTCT-3′






The 3′ PCR primer contained an EcoRI site with the following sequence:








(SEQ. ID. NO.:139)









5′-TCTGAATTCAAAGATGATGGTCTCAGCGGTGCC-3′-.






The second round of PCR utilized pituitary DNA as a template and a 5′ PCR primer contained a HindIII site with the following sequence:








(SEQ. ID. NO.:140)









5′-GCAAAGCTTGCTCATGGATTCTGGGCCTCTGTGGG-3′






and the 3′ PCR primer contained an EcoRI site with the following sequence:








(SEQ. ID. NO.:141)









5′-TCTGAATTCAAAGATGATGGTCTCAGCGGTGCCTTCCC-3′.






The resulting PCR fragment was digested with HindIII and EcoRI restriction site and cloned into a HindIII-EcoRI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
4. 5HT6
The endogenous human 5HT6 receptor was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. Both rounds of PCR had the following cycle condition: 30 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min and 45 sec. The first round of PCR utilized a caudate nucleus DNA, as a template, and a 5′ PCR primer that contained a HindIII site with the following sequence:







(SEQ. ID. NO.:142)









5′-CATAAGCTTTCCCGCCACCCTATCACT-3′






The 3′ PCR primer contained an EcoRI site with the following sequence:








(SEQ. ID. NO.:143)









5′-ACTGAATTCTGCTCAATCCAGCTCCCCA-3′-.






The second round of PCR also utilized caudate nucleus DNA as a template and a 5′ PCR primer that contained an EcoRV site with the following sequence:








(SEQ. ID. NO.:144)









5′-CCTCGGATATCATGGTCCCAGAGCCGGGCCC-3′






and a 3′ PCR primer that contained a XbaI site with the following sequence:








(SEQ. ID. NO.:145)









5′-CAGCTCTAGATTGGCCAGCCCCAAGCCCGGGT-3′.






Nucleic acid and amino acid sequences were thereafter determined and verified.
5. Dopamine D1
Dopamine D1 was subcloned from a full length cDNA clone obtained from the American Type Culture Collection.
6. Dopamine D2
Dopamine D2 was subcloned from a full length cDNA clone obtained from the American Type Culture Collection.
7. Dopamine D5
To obtain Dopamine D5, PCR was performed using genomic cDNA as template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides.
Dopamine D5 receptor contained no intron in the coding region. However, Dopamine D5 receptor contained two pseudogenes with 8 bp fame shift insertion within the coding region. In order to avoid the pseudogenes, the DNA fragment 5′ and 3′ of the frame shift insert was each amplified from genomic DNA. The 5′ PCR fragment was obtained utilizing the following primer set:









(SEQ. ID. NO.:146; sense)



′-CCTGAATTCCAGCCCGAAATGCTGCCGCCAG-3′



 



(SEQ. ID. NO.:147; antisense)



5′-GGTCCACGCTGATGACGCACAGGTTC-3′.



 



and






3′ PCR fragment was obtained utilizing the following primer set:










(SEQ. ID. NO.:148; sense)



5′-GAACCTGTGCGTCATCAGCGTGGACC-3′



 



(SEQ. ID. NO.:149; antisense)



5′-TGCGGATCCATGAGGGGGTTTCTTAATG-3′.






The 5′ and 3′ PCR fragments were then used as co-templates to obtain the full length cDNA using the pfu polymerase and SEQ.ID.NO.:146 and SEQ.ID.NO.:149 as primers. The cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. for 2 min 30 sec and 72° C. for 1 min 30 sec.

The resulting PCR fragment was digested with EcoRI and BamHI restriction sites and cloned into an EcoRI-BamHI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
8. ETA
The endogenous human ETA was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 2 sec. The 5′ PCR fragment, containing a HindIII site, was obtained utilizing genomic DNA, as a template, and the following primer set:







(SEQ. ID. NO.:150)









5′-CGGAAGCTTCTGGAGCAGGTAGCAGCATG-3′



 



and



 







(SEQ. ID. NO.:151)









5′-TGGGCAATAGTTGTGCATTGAGCCA-3′-.






The 3′ PCR fragment, containing BamHI site, was obtained by pfu polymerase (Stratagene) using IMAGE 666747, as a template, and the following primer set:








(SEQ. ID. NO.:152)









5′-CTAATTTGGTCCTACCCAGCAATGGC-3′



 



and



 







(SEQ. ID. NO.:153)









5′-CTTGGATCCAGATGAGCTGTATTTATTACTGGAACG-3′.






The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 64° C. for 1 min and 72° C. for 2 sec. The 5′ and 3′ PCR fragments were then used as co-templates to obtain the full length cDNA using the pfu polymerase (Stratagene) and the following primers:








(SEQ. ID. NO.:154)









5′-AATAAGCTTCAAGATGGAAACCCTTTGCCTCAG-3′



 







(SEQ. ID. NO.:155)









5′-CGTTCATGCTGTCCTTATGGCTGCTC-3′.






The PCR cycle condition for the full length clone was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 2 min 10 sec.

The resulting PCR fragment was digested with EcoRI restriction site and cloned into an EcoRI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
9. mGluR1
The endogenous human mGluR1 was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer. The cycle condition for the first round of PCR was as follows: 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 50 sec. The 5′ PCR fragment contained a SalI site and was obtained utilizing hippocampus DNA as a template, and the following primer set:







(SEQ. ID. NO.:156)









5′-GCAGGCTGTCGACCTCGTCCTCACCACCATGGTC-3′



 



and



 







(SEQ. ID. NO.:157)









5′-AATGGGCTCACAGCCTGTTAGATCTGCATTGGGCCAC-3′-.






The middle PCR fragment was obtained utilizing genomic DNA as a template, where the cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 50 sec, with the following primer set:







(SEQ. ID. NO.:158)









5′-TAACAGGCTGTGAGCCCATTCCTGTGCG-3′



 



and



 







(SEQ.ID.NO.:159)









5′-TTAGAATTCGCATTCCCTGCCCCTGCCTTCTTTC-3′.






The 3′ PCR fragment contained a BamHI site and was obtained utilizing genomic cDNA, as a template, and the following primer set:







(SEQ. ID. NO.:160)









5′-TGCGAATTCTAATGGCAAGTCTGTGTCATGGTC-3′



 



and



 







(SEQ. ID. NO.:161)









5′-TCCGGATCCCAGGGTGGAAGAGCTTTGCTTGTA-3′.






The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 15 sec.

The resulting PCR fragment was digested with SalI and BamHI restriction site and cloned into a SalI-BamHI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
10. GPR24 (also known as MCH or SLC-1)
The endogenous human GPR24 was obtained by PCR using genomic DNA as template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 56° C. for 1 min and 72° C. for 1 min and 20 sec. The 5′ PCR primer contained a HindIII site with the sequence:







(SEQ. ID. NO.:162)









5′-GTGAAGCTTGCCTCTGGTGCCTGCAGGAGG-3′






and the 3′ primer contained an EcoRI site with the sequence:








(SEQ. ID. NO.:163)









5′-GCAGAATTCCCGGTGGCGTGTTGTGGTGCCC-3′.






The 1.3 kb PCR fragment was digested with HindIII and EcoRI and cloned into HindIII-EcoRI site of CMVp expression vector. Later the cloning work by Lakaye et al showed that there is an intron in the coding region of the gene. Thus the 5′ end of the cDNA was obtained by 5′ RACE PCR using Clontech's marathon-ready hypothalamus cDNA as template and the manufacturer's recommended protocol for cycling condition. The 5′ RACE PCR for the first and second round PCR were as follows:








5′-CATGAGCTGGTGGATCATGAAGGG-3′
(SEQ. ID. NO.:164)


 


and


 


5′-ATGAAGGGCATGCCCAGGAGAAAG-3′.
(SEQ. ID. NO.:165)






Nucleic acid and amino acid sequences were thereafter determined and verified.
11. NTSR1
The endogenous human NTSR1 was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min and 10 sec. The 5′ PCR fragment, containing a HindIII site, was obtained utilizing genomic DNA, as a template, and the following primer set:







(SEQ. ID. NO.:166)









5′-CCCAAGCTTCCAGCCCCGGAGGCGCCGGAC-3′



 



and



 







(SEQ. ID. NO.:167)









5′-TGAAGGTGTTGACCTGTATGACGACCTTGACGGTGGG-3′-.






The 3′ PCR fragment, containing an EcoRI site, was obtained utilizing brain cDNA, as a template, and the following primer set:








(SEQ. ID. NO.:168)









5′-GGTCGTCATACAGGTCAACACCTTCATGTCCTTCATA-3′



 



and



 







(SEQ. ID. NO.:169)









5′-CACGAATTCGTACAGCGTCTCGCGGGTGGCATT-3′.






The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min and 10 sec. The 5′ and 3′ PCR fragments were then used as co-templates to obtain the full length cDNA using the pfu polymerase (Stratagene) and









5′-ATCACCATGCGCCTCAACAGCTCCGC-3′
(SEQ. ID. NO.:170)






and SEQ.ID.NO.:169 as primers. The cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 2 min 10 sec.

The resulting PCR fragment was digested with HindIII and EcoRI restriction site and cloned into a HindIII-EcoRI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
12. NTSR2
The endogenous human NTSR2 was obtained by PCR using a template and pfu polymerase (Stratagene) with the buffer system provided by the manufacturer. The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 15 sec. The 5′ PCR fragment was obtained utilizing IMAGE 1537523, as a template, and the following primer set:







(SEQ. ID. NO.:171)









5′-ATCACCATGGAAACCAGCAGCCCGCGGC-3′



 



and



 







(SEQ. ID. NO.:172)









5′-CGGGGTAGAAGTGGACGGCACTTGGG-3′-.






The 3′ PCR fragment, containing an EcoRI site, was obtained utilizing caudate nucleus cDNA, as a template, and the following primer set:








(SEQ. ID. NO.:173)









5′-GCTCCCAAGTGCCGTCCACTTCTACC-3′



 



and



 







(SEQ. ID. NO.:174)









5′-TTAGAATTCGGTCCGGGTTTCTGGGGGATCC-3′.






The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min and 20 sec. The 5′ and 3′ PCR fragments were then used as co-templates to obtain the full length cDNA using the pfu polymerase (Stratagene) and SEQ.ID.NO.:171 and SEQ.ID.NO.:174 as primers. The PCR cycle condition for the full length clone was 30 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 2 min 10 sec.

The resulting PCR fragment was digested with EcoRI restriction site and cloned into an EcoRI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
13. OPRM1
The endogenous human OPRM1 was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer. The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 40 sec. The 5′ PCR fragment, containing a HindIII site, was obtained utilizing genomic DNA as a template, and the following primer set:







(SEQ. ID. NO.:175)







5′-CCCAAGCTTCAGTACCATGGACAGCAGCGCTGCC-3′


 


and


 







(SEQ. ID. NO.:176)







5′-CATCTTGGTGTATCTGACAATCACATACATGACCAGGAA-3′-.






The middle PCR fragment was obtained utilizing genomic DNA as a template, where the cycle condition was 30 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 40 sec, and the following primer set:







(SEQ. ID. NO.:177)







5′-GTATGTGATTGTCAGATACACCAAGATGAAGACTGCCAC-3′


 


and


 







(SEQ. ID. NO.:178)







5′-TACAATCTATGGAACCTTGCCTGTATTTTGTTGTAGCCA-3′.






The 3′ PCR fragment was obtained utilizing brain cDNA, as a template, and the following primer set:







(SEQ. ID. NO.:179)









5′-CAAAATACAGGCAAGGTTCCATAGATTGTACACTAACAT-3′



and



 







(SEQ. ID. NO.:180)









5′-CGGGCAACGGAGCAGTTTCTGCTTCAG-3′.






The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 63° C. for 1 min and 72° C. for 1 min and 15 sec.

The 5′PCR fragment and the middle PCR fragment were used as templates to obtain the 5′-region through the middle region of OPRM1 (“5′-middle PCR fragment”) using the pfu polymerase (Stratagene) and SEQ.ID.NO.:175 and SEQ.ID.NO.:178 with the cycle conditions as follows: 30 cycles of 94° C. for 1 min, 63° C. for 1 min and 72° C. for 1 min and 15 sec.
The 5′-middle PCR fragment and 3′ PCR fragment were then used as templates to obtain the full length cDNA using the pfu polymerase (Stratagene) and SEQ.ID.NO.:175 and SEQ.ID.NO.:180 as primers. The cycle condition for the full length PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min 15 sec.
The resulting PCR fragment was digested with HindIII restriction site and cloned into a HindIII pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
14. OPRM1A
The endogenous human OPRM1A was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer. The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 40 sec. The 5′ PCR fragment, containing a HindIII site, was obtained utilizing genomic DNA as a template, and SEQ.ID.NO.:175 and SEQ.ID.NO.:176.
The middle PCR fragment was obtained utilizing genomic DNA as a template, where the cycle condition was 30 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 40 sec, and SEQ.ID.NO.:177 and SEQ.ID.NO.:178.
The 3′ PCR fragment was obtained utilizing genomic DNA as a template, and SEQ.ID.NO.:179 and SEQ.ID.NO.:180. The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min and 30 sec.
The 5′PCR fragment and the middle PCR fragment were used as co-templates to obtain the 5′-region through the middle region of OPRM1A (“5′-middle PCR fragment”) using the pfu polymerase (Stratagene) and SEQ.ID.NO.:175 and SEQ.ID.NO.:178 with the cycle conditions as follows: 30 cycles of 94° C. for 1 min, 63° C. for 1 min and 72° C. for 1 min and 15 sec.
The 5′-middle PCR fragment and 3′ PCR fragment were then used as co-templates to obtain the full length cDNA using the pfu polymerase (Stratagene) and SEQ.ID.NO.:175 and SEQ.ID.NO.:180 as primers. The cycle condition for the full length PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min 15 sec.
The resulting PCR fragment was digested with HindIII restriction site and cloned into a HindIII pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
15. OX1R
The OX1R EST clone 40608 is a full length cDNA clone. However, it contained a 4 bp frame shift insertion. To remove the insert, the fragments 5′ and 3′ of the frame shift insert was each obtained by PCR using EST clone 40608 as template and two primer pairs. The 5′ primer set, containing an EcoRI site, were as follows:









(SEQ. ID.NO.:181; sense)



5′-ATGGAATTCTGCTGCAGCGGCTCCTGAGCTC-3′



 



(SEQ. ID. NO.:182; antisense)



5′-ACGGACACAGCCTGTAGATAGGGGATGACCTTGCAG-3′






and the 3′ primer set, containing a BamHI site, were as follows:










(SEQ. ID. NO.:183; sense)



5′-ATCCCCTATCTACAGGCTGTGTCCGTGTCAGTGGCAG-3′



 



(SEQ. ID. NO.:184; antisense).



5′-GGAGGATCCAGGGCAGCCCTCGCTGAGGGC-3′.






The 5′ and 3′ PCR fragments were then used as co-templates to obtain the full length cDNA using SEQ.ID.NO.:181 and SEQ.ID.NO.:184 as primers. The cycle condition for each PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. for 2 min 30 sec and 72° C. for 1 min 30 sec.

The resulting PCR fragment was digested with EcoRI and BamHI restriction sites and cloned into an EcoRI-BamHI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
16. PTHR1
The endogenous human PTHR1 was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer. The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 30 sec. The 5′ PCR fragment, containing a HindIII site, was obtained utilizing kidney cDNA, as a template, and the following primer set:







(SEQ. ID. NO.:185)









5′-CGCAAGCTTAGGCGGTGGCGATGGGGACCGCC-3′



 



and



 







(SEQ. ID. NO.:186)









5′-GGATGTGGTCCCATTCCGGCAGACAG-3′-.






The 3′ PCR fragment, containing an EcoRI site, was obtained by pfu PCR (Stratagene) and IMAGE 1624048, as a template, and the following primer set:







(SEQ. ID. NO.:187)









5′-AGGAGGCACCCACTGGCAGCAGGTA-3′



 



and



 







(SEQ. ID. NO.:188)









5′-GCCGAATTCCATGACTGTCTCCCACTCTTCCTG-3′.






The cycle condition for 3′ PCR reaction was as follows: 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 30 sec. The 5′ and 3′ PCR fragments were then used as co-templates to obtain the full length cDNA using the pfu polymerase (Stratagene) and SEQ.ID.NO.:185 and SEQ.ID.NO.:188 as primers. The PCR cycle condition for the full length clone was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 3 sec.

The resulting PCR fragment was digested with HindIII and EcoRI restriction site and cloned into a HindIII-EcoRI pCMV expression vector. Nucleic acid and amino acid sequences were thereafter determined and verified.
17. SST2
SST2 was obtained by subcloning EST 06818 into a pCMV vector.
Table E below indicates the GenBank Accession number for which the endogenous receptors set forth above can be located, and for which the endogenous nucleic and amino acid sequences are provided.








TABLE E





Receptor Identifier
GenBank Accession Number







5HT-1A
X13556


5HT-1B
D10995


5HT-1D
M81589


5HT-1E
M91467


5HT-1F
L04962


5HT-2B
X77307


5HT-4A
Y08756


5HT-4B
Y12505


5HT-4C
Y12506


5HT-4D
Y12507


5HT-4E
AJ011371


5HT-5A
X81411


5HT6
L41147


5HT7
L21195


AVPR1A
AF030625


AVPR1B
D31833


AVPR2
NM_000054


BBR3
X76498


BDKR1
AJ238044


BDKR2
NM_000623


C3a
U62027


C5a
M62505


CB1
X54937


CB2
X74328


CCR2b
U03882


CCR3
U28694


CCR5
U54994


CCR8
U45983


CCR9
AJ132337


CRFR1
L23332


CXCR4
AJ224869


Dopamine D1
X55758


Dopamine D2
S62137


Dopamine D3
U32499


Dopamine D5
M67439


ETA
X61950


ETB
L06623


FPR1
M60627


FPRL1
M76672


GALR1
L34339


GALR2
AF040630


GALR3
AF073799


GIP
U39231


mGluR1
L76627


GPR5
L36149


GPR24 (also known as MCH or SLC-1)
U71092


GRPR
M73481


M1
X15263


M2
X15264


M3
X15266


M4
X15265


M5
M80333


MC3
L06155


NK1R
M74290


NK2R
M57414


NK3R
M89473


NMBR
M73482


NPY5
U94320


NTSR1
X70070


NTSR2
Y10148


OPRD
U07882


OPRL1
X77130


OPRK
U11053


OPRM
L25119


OPRM1A
L25119


OX1R
AF041243


OX2R
AF041245


PACAP
D17516


PAF
S56396


PGE EP1
L22647


PGE EP2
U19487


PGE EP4
NM_000958


PTHR1
L04308


PTHR2
U25128


SCTR
U28281


SST1
M81829


SST2
M81830


SST3
M96738


SST4
D16826


SST5
D16827


TSHR
AF035261


VIPR
L13288


VIPR2
X95097









Example 2
Preparation of Non-Endogenous, Versions of the known GPCRs
A. Site-Directed Mutagenesis
Those skilled in the art are credited with the ability to select techniques for mutation of a nucleic acid sequence. Presented below are approaches utilized to create non-endogenous versions of several of the human GPCRs disclosed above. The mutations disclosed below are based upon an algorithmic approach whereby the 16th amino acid (located in the IC3 region of the GPCR) from a conserved proline residue (located in the TM6 region of the GPCR, near the TM6/IC3 interface) is mutated, most preferably to a lysine amino acid residue.
In most of the examples of this Example 2, the algorithmic approach set forth above was used to identify the amino acid residue to be mutated. However, several GPCRs set forth below utilized a modified algorithmic approach (e.g., CRFR1, GIP, mGluR1, GPR24, PTHR1, PTHR2, SCTR, TSHR, VIPR and VIPR2). This modified approach focuses on a conserved proline residue (also located in the TM6 region of the GPCR, near the TM6/IC3 interface) whereby the 5th amino acid upstream from the proline is generally, but not always, a threonine residue. For these receptors, the endogenous 5th amino acid residue is mutated, most preferably to a proline amino acid residue.
Other mutation approaches can be used (e.g., mGluR1, GPR24 and TSHR) and one skilled in the art is credited with the ability to select techniques for mutation of a nucleic acid sequence. The importance here is that the mutation leads to a constitutively activated receptor and given the extension approaches set forth herein for determination of constitutively activity, routine analysis can be employed in this context.
Preparation of non-endogenous known GPCRs is preferably accomplished by using TRANSFORMER SITE-DIRECTEDTM Mutagenesis Kit (Stratagene, according to manufacturer's instructions) or QUIKCHANGE SITE-DIRECTEDTM Mutagenesis (Clontech). Endogenous GPCR is preferably used as a template and two mutagenesis primers utilized, as well as, most preferably, a lysine mutagenesis oligonucleotide and a selection marker oligonucleotide (SEQ.ID.NO.: 252; included in Stratagene's kit). For convenience, the codon mutation incorporated into the known GPCR and the respective oligonucleotides are noted, in standard form (Table F):










TABLE F







5′-3′ orientation (sense),



Receptor
Codon
(SEQ. ID. NO.) mutation
5′-3′ orientation (antisense)


Identifier
Mutation
underlined
(SEQ. ID. NO.)







5HT-1A
V343K
CGAGAGAGGAAGACAAAG
ATGCCCAGCGTCTTCTTT




AAGACGCTGGGCAT (189)
GTCTTCCTCTCTCG (190)


 


5HT-1B
T313K
GGGAGCGCAAAGCCAAGA
GATCCCTAGGGTCTTCTT




AGACCCTAGGGATC (191)
GGCTTTGCGCTCCC (192)


 


5HT-1D
T300K
CGAGAAAGGAAAGCCAAG
GAATGATGCCCAGGATT




AAAATCCTGGGCATCATTC
TTCTTGGCTTTCCTTTCT




(193)
CG (194)


 


5HT-1E
A290K
AGGGAACGGAAGGCAAAA
AGCCCCAGGATGCGTTT




CGCATCCTGGGGCT (195)
TGCCTTCCGTTCCCT





(196)


 


5HT-1F
A292K
CAAGAGAACGGAAAGCAA
GATTAATCCCAGGGTAG




AGACTACCCTGGGATTAAT
TCTTTGCTTTCCGTTCTC




C (197)
TTG (198)


 


5HT-2B
S323K
AACGAACAGAGAGCCAAA
CAATCCCTAGGACCTTTT




AAGGTCCTAGGGATTG
TGGCTCTCTGTTCGTT




(199)
(200)


 


5HT-4A
A258K
GGACAGAGACCAAAGCAA
GATGCACAGGGTCTTCTT




AGAAGACCCTGTGCATC
TGCTTTGGTCTCTGTCC




(201)
(202)


 


5HT-4B
A258K
GGACAGAGACCAAAGCAA
GATGCACAGGGTCTTCTT




AGAAGACCCTGTGCATC
TGCTTTGGTCTCTGTCC




(203)
(204)


 


5HT-4C
A258K
GGACAGAGACCAAAGCAA
GATGCACAGGGTCTTCTT




AGAAGACCCTGTGCATC
TGCTTTGGTCTCTGTCC




(205)
(206)


 


5HT-4D
A258K
GGACAGAGACCAAAGCAA
GATGCACAGGGTCTTCTT




AGAAGACCCTGTGCATC
TGCTTTGGTCTCTGTCC




(207)
(208)


 


5HT-4E
A258K
GGACAGAGACCAAAGCAA
GATGCACAGGGTCTTCTT




AGAAGACCCTGTGCATC
TGCTTTGGTCTCTGTCC




(209)
(210)


 


5HT-5A
A284K
AAGGAGCAGCGGGCCAAG
GATGCCCACCATGAGCT




CTCATGGTGGGCATC (211)
TGGCCCGCTGCTCCTT





(212)


 


5HT-6
S267K
CTGAAGGCCAAGCTTACGCT
ATGCCCAGCGTAAGCTTG




GGGCATCCTGCTGGGCA
GCCTTCAGGGCCTTCCTG




(213)
CT (214)


 


5HT-7
A326K
GAACAGAAAGCAAAGACCA
CCCAGGGTGGTCTTTGCT




CCCTGGGGATCATCGT (215)
TTCTGTTCTCGCTTAAA





(216)


 


AVPR1A
V290K
GCCAAGATCCGCACGAAGA
CGATCACAAAAGTCATCT




AGATGACTTTTGTGATCG
TCTTCGTGCGGATCTTGG




(217)
C (218)


 


AVPR1B
V280K
GGCCAAGATCCGAACAAAG
CGATGACAAAGGTCATCT




AAGATGACCTTTGTCATC
TCTTTGTTCGGATCTTGG




(219)
CC (220)


 


AVPR2
V270K
GCTGTGGCCAAGACTAAGA
CACTAGCGTCATCCTCTT




GGATGACGCTAGTG (221)
AGTCTTGGCCACAGC





(222)


 


BBR3
270K
CGAAAGAGAATTAAAAGAA
AATACCGTTCTTTTAATT




CGGTATTGGTGTTG (223)
CTCTTTCGGGATTC (224)


 


BDKR1
T249K
GCCGCAAGGATAGCAAGAC
GTGAGGATCAGCGCTTTG




CAAAGCGCTGATCCTCAC
GTCTTGCTATCCTTGCGG




(225)
C (226)


 


BDKR2
T269K
CGGAGAGGAGGGCCAAGGT
ACCAGGACTAGCACCTTG




GCTAGTCCTGGT (227)
GCCCTCCTCTCCG (228)


 


C3a
F376K
CGCCAAGTCTCAGAGCAAA
CACCACGGCCACTCGCTT




ACCAAGCGAGTGGCCGTGG
GGTTTTGCTCTGAGACTT




TG (229)
GGCG (230)


 


C5a
L241K
CGGTCCACCAAGACAAAGA
TGCCACCACCACCTTCTT




AGGTGGTGGTGGCA (231)
TGTCTTGGTGGACCG





(232)


 


CB1
A342K
CGCATGGACATTAGGTTAA
TCAGGACCAGGGTCTTCT




AGAAGACCCTGGTCCTGA
TTAACCTAATGTCCATGC




(233)
G (234)


 


CB2
A244K
GGCTGGATGTGAGGTTGAA
CACTAGCCCTAGGGTCTT




GAAGACCCTAGGGCTAGTG
CTTCAACCTCACATCCAG




(235)
CC (236)


 


CCR2b
V242K
GAAGAGGCATAGGGCAAAG
GGTGAAGATGACTCTCTT




AGAGTCATCTTCACC (237)
TGCCCTATGCCTCTTC





(238)


 


CCR3
I238K
GTAAAAAAAAGTACAAGGC
GATGACAAAAATGAGCC




CAAGCGGCTCATTTTTGTCA
GCTTGGCCTTGTACTTTTT




TC (239)
TTTAC (240)


 


CCR5
V234K
GAAGAGGCACAGGGCTAAG
GATGGTGAAGATAAGCC




AGGCTTATCTTCACCATC
TCTTAGCCCTGTGCCTCT




(241)
TC (242)


 


CCR8
I237K
CCACAACAAGACCAAGGCC
CCACAATGAGCACCAAC




AAGAGGTTGGTGCTCATTGT
CTCTTGGCCTTGGTCTTG




GG (243)
TTGTGG (244)


 


CCR9
L253K
TCCAAGCACAAAGCCAAAA
GGACAGTGATGGTCACTT




AAGTGACCATCACTGTCC
TTTTGGCTTTGTGCTTGG




(245)
A (246)


 


CRFR1
T316P
GAAGGCTGTGAAAGCCCCT
GCAGCAGCACCAGAGGG




CTGGTGCTGCTGC (247)
GCTTTCACAGCCTTC (248)


 


CXCR4
L238K
AGAAGCGCAAGGCCAAGAA
TGAGGATGACTGTGGTCT




GACCACAGTCATCCTCA
TCTTGGCCTTGCGCTTCT




(249)
(250)


 


Dopamine D1
L271K
GAGAAACTAAAGTCAAGAA
CTCCTTCGGTCCTCCTAT




GACTCTGTG (251)
CGTTGTCAGAAGT (252)


 


Dopamine D2
T372K
GAGAAGAAAGCCAATCAGA
GGCGAGCATCTGAGTGG




TGCTCGCC (253)
CTTTCTTCTC (254)


 


Dopamine D3
T328K
GAGAAGAAGGCAAAACAAA
GGCCACCATTTGTTTTGC




TGGTGGCC (255)
CTTCTTCTC (256)


 


Dopamine D5
L295K
AAGAAGGAGACCAAAGTTA
CGACAGGGTCTTTTTAAC




AAAAGACCCTGTCG (257)
TTTGGTCTCCTTCTT (258)


 


ETA
A305K
CAGCGTCGAGAAGTGAAAA
TACAACCAAGCAGAAAA




AAACAGTTTTCTGCTTGGTT
CTGTTTTTTTCACTTCTCG




GTA (259)
ACGCTG (260)


 


ETB
A322K
CAGAGACGGGAAGTGAAGA
CCAGGCAAAAGACGGTT




AAACCGTCTTTTGCCTGG
TTCTTCACTTCCCGTCTCT




(261)
G (262)


 


FPR1
L240K
AAGTCCAGTCGTCCCAAAC
AAGGAGAGGACCCGTTT




GGGTCCTCTCCTT (263)
GGGACGACTGGACTT





(264)


 


FPRL1
L240K
AAATCCAGCCGTCCCAAAC
GCAGTGAGGACCCGTTTG




GGGTCCTCACTGC (265)
GGACGGCTGGATTT (266)


 


GALR1
A246K
CCAAGAAAAAGACTAAACA
CTCCTTCGGTCCTCCTAT




GACAGTTCTGG (267)
CGTTGTCAGAAGT (252)


 


GALR2
T235K
GCCAAGCGCAAGGTGAAAC
GAGGATCATGCGTTTCAC




GCATGATCCTC (268)
CTTGCGCTTGGCG (269)


 


GIP
T343P
AGGCTGGCTCGCTCCCCGCT
GCACCAGCGTCAGCGGG




GACGCTGGTGC (270)
GAGCGAGCCAGCCT (271)


 


mGluR1
3′ Deletion
See alternative approach
See alternative approach




below
below


 


GPR5
V224K
CGGCGCCACCGCACGAAAA
CTCCTTCGGTCCTCCTAT




AGCTCATCTTC (272)
CGTTGTCAGAAGT (252)


 


GPR24 (also
T255K
See alternative approach
See alternative approach


known as MCH
T255K/T257R
below
below


or SLC-1)
24-IC3-SST2



C305Y



P271L



W269C



W269F



W269L



F265I



I261Q



D140N


 


GRPR
A263K
GGAAGCGACTTAAGAAGAC
CAGCACTGTCTTCTTAAG




AGTGCTGGTGTTT (273)
TCGCTTCCGGGATTC (274)


 


M1
A364K
AAGGAGAAGAAGGCGAAAC
GGCACTCAGGGTCCGTTT




GGACCCTGAGTGCC (275)
CGCCTTCTTCTCCTT (276)


 


M2
T386K
CCGGGAAAAGAAAGTCAAG
AGCCAAGATTGTCCTCTT




AGGACAATCTTGGCT (277)
GACTTTCTTTTCCCGG





(278)


 


M3
A490K
GGTCAAGGAGAAGAAAGCG
CGCACTGAGGGTCTGTTT




AAACAGACCCTCAGTGCG
CGCTTTCTTCTCCTTGACC




(279)
(280)


 


M4
T399K
GGGAGCGCAAAGTGAAACG
GGCAAAGATCGTTCGTTT




AACGATCTTTGCC (281)
CACTTTGCGCTCCC (282)


 


M5
A441K
GTCAAAGAGAGGAAAGCAA
GGCACTCAGTGTCTGTTT




AACAGACACTGAGTGCC
TGCTTTCCTCTCTTTGAC




(283)
(284)


 


MC3
A241K
GCAACACTCATGTATGAAG
CTCCTTCGGTCCTGCTAT




GGGAAAGTCACCATCACC
CGTTGTCAGAAGT (252)




(285)


 


NK1R
V247K
GCCAAGCGCAAGGTGAAGA
GACAATCATCATTTTCTT




AAATGATGATTGTC (286)
CACCTTGCGCTTGGC





(287)


 


NK2R
V249K
GCCAAGAAGAAGTTTAAGA
AGCACCATGGTCTTCTTA




AGACCATGGTGCT (288)
AACTTCTTCTTGGC (289)


 


NK3R
V298K
GGCCAAAAGAAAGGTTAAG
CAATAATCATCATTTTCT




AAAATGATGATTATTG (290)
TAACCTTTCTTTTGGCC





(291)


 


NMBR
A265K
CACGGAAACGCCTGAAAAA
CAAGCACAATTTTTTTCA




AATTGTGCTTG (292)
GGCGTTTCCGTG (293)


 


NPY5
F367K
GAATAAAAAAGAGATCACG
GGTCAGTCTGTACTTAAC




AAGTGTTAAGTACAGACTG
ACTTCGTGATCTCTTTTTT




ACC (294)
AT (295)


 


NTSR1
V302K
GCCCTGCGGCACGGCAAGC
GCACGTAGGACGCGCTTG




GCGTCCTACGTGC (296)
CCGTGCCGCAGGGC (297)


 


NTSR2
V269K
AGCCTCCAGCGCAGCAAGC
GGCTCTGAGAACCTGCTT




AGGTTCTCAGAGCC (298)
GCTGCGCTGGAGGCT





(299)


 


OPRD
T260K
GCCTGCGGCGCATCAAGCG
ACCAGCACCATGCGCTTG




CATGGTGCTGGT (300)
ATGCGCCGCAGGC (301)


 


OPRL1
T262K
ACCTGCGGCGCATCAAGCG
CACCAGCACCAGCCGCTT




GCTGGTGCTGGTG (302)
GATGCGCCGCAGGT (303)


 


OPRK
T273K
ACCTGCGTAGGATCAAGAG
CACCAGGACCAGTCTCTT




ACTGGTCCTGGTG (304)
GATCCTACGCAGGT (305)


 


OPRM
T281K
GGGAATCTTCGAAGGATCA
CACCAGCACCATCCTCTT




AGAGGATGGTGCTGGTG
GATCCTTCGAAGATTCC




(306)
(307)


 


OPRM1A
T281K
GGGAATCTTCGAAGGATCA
CACCAGCACCATCCTCTT




AGAGGATGGTGCTGGTG
GATCCTTCGAAGATTCC




(308)
(309)


 


OX1R
A297K
CGGAGGAAGACAAAAAAGA
CCATCAGCATCTTTTTTG




TGCTGATGG (310)
TCTTCCTCCG (311)


 


OX2R
A303K
CCAGAAGGAAAACAAAACG
CTCCTTCGGTCCTCCTAT




GATGTTGATG (312)
CGTTGTCAGAAGT (252)


 


PACAP
T355K
CGACTGGCCCGGTCCCCCCT
GGATGAGCAGCAGGGGG




GCTGCTCATCC (313)
GACCGGGCCAGTCG (314)


 


PAF
L231K
GTCAAGCGCCGGGCGAAGT
GTGCACACCATCCACTTC




GGATGGTGTGCAC (315)
GCCCGGCGCTTGAC (316)


 


PGE EP1
V296K
CACGACGTGGAGATGAAGG
CCGACAAGCTGGCCCTTC




GCCAGCTTGTCGG (317)
ATCTCCACGTCGTG (318)


 


PGE EP2
L263K
GAGGAGACGGACCACAAGA
CATGATAGCCAGGAGAA




TTCTCCTGGCTATCATG (319)
TCTTGTGGTCCGTCTCCT





C (320)


 


PGE EP4
V271K
GCCGAGATCCAGATGAAGA
GGTGGCAATGAGTAAGA




TCTTACTCATTGCCACC (321)
TCTTCATCTGGATCTCGG





C (322)


 


PTHR1
T410P
GGAAGCTGCTCAAATCCCC
GCATGAGCACCAGCGGG




GCTGGTGCTCATGC (323)
GATTTGAGCAGCTTCC





(324)


 


PTHR2
T365P
GGAAACTGGCCAAATCGCC
GGACCAGGACCAGTGGC




ACTGGTCCTGGTCC (325)
GATTTGGCCAGTTTCC





(326)


 


SCTR
T344P
CGCCTGGCCAGGTCCCCTCT
GGATCAGCAGGAGAGGG




CCTGCTGATCC (327)
GACCTGGCCAGGCG (328)


 


SST1
T270K
CGAGCGCAAGATCAAATTA
CTCCTTCGGTCCTCCTAT




ATGGTGATGG (329)
CGTTGTCAGAAGT (252)


 


SST2
T255K
AGAAGAAGGTCAAACGAAT
GGACACCATTCGTTTGAC




GGTGTCCATCGTG (330)
CTTCTTCTCAGACT (331)


 


SST3
T256K
GAACGCAGGGTCAAGCGCA
CTCCTTCGGTCCTCCTAT




TGGTGGTGGCC (332)
CGTTGTCAGAAGT (252)


 


SST4
T258K
CGGAGAAGAAAATCAAAAG
CTCCTTCGGTCCTCCTAT




GCTGGTGCTG (333)
CGTTGTCAGAAGT (252)


 


SST5
T247K
TCGGAGCGAAAGGTGAAGC
ACCATGCGCTTCACCTTT




GCATGGTGTTGGTGGT (334)
CGCTCCGAGCGCCGCCG





(335)


 


TSHR
V509A
See alternative approach
See alternative approach



D619G
below
below



A623I



A623K



C672Y



D619G/A623K



V509A/C672Y



V509A/A623K/C672Y


 


VIPR
T343P
AGGCTAGCCAGGTCCCCACT
GGATCAGCAGGAGTGGG




CCTGCTGATCC (336)
GACCTGGCTAGCCT (337)


 


VIPR2
T330P
AGGCTGGCCAAGTCCCCGCT
GGATAAGCAGGAGCGGG




CCTGCTTATCC (338)
GACTTGGCCAGCCT (339)









B. Alternative Approaches to Mutation
1. mGluR1
Preparation of a non-endogenous version of the human mGluR1 receptor was accomplished by deleting a portion of the intracellular region at the 3′ end. The non-endogenous human mGluR1 was obtained by PCR using a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer. The cycle condition for the first round of PCR was as follows: 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 50 sec. The 5′ PCR fragment contained a SalI site and was obtained utilizing hippocampus DNA as a template, and the following primer set:







(SEQ. ID. NO.:340)









5′-GCAGGCTGTCGACCTCGTCCTCACCACCATGGTC-3′



 



and



 







(SEQ. ID. NO.:341)









5′-AATGGGCTCACAGCCTGTTAGATCTGCATTGGGCCAC-3′-.






The middle PCR fragment was obtained utilizing genomic DNA as a template, where the cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 50 sec, with the following primer set:







(SEQ. ID. NO.:342)









5′-TAACAGGCTGTGAGCCCATTCCTGTGCG-3′



 



and



 







(SEQ. ID. NO.:343)









5′-TTAGAATTCGCATTCCCTGCCCCTGCCTTCTTTC-3′.






The 3′ PCR fragment was obtained by utilizing the endogenous mGluR1 clone as a co-template to obtain the full length cDNA using the pfu polymerase. The cycle condition for this PCR reaction was 30 cycles of 94° C. for 1 min, 65° C. for 2 min 30 sec and 72° C. for 1 min 30 sec. and the following primer set:







(SEQ. ID. NO.:344)









5′-TGCGAATTCTAATGGCAAGTCTGTGTCATGGTC-3′



 



and



 







(SEQ. ID. NO.:345)









5′-TGCGGATCCTCTTCGGAAGATGTTGAGGAAAGTG-3′.






(See, SEQ.ID.NO.:346 for nucleic acid sequence and SEQ.ID.NO.:347 for amino acid sequence).

2. GPR24 (MCH or SLC-1)
Preparation of non-endogenous versions of the human GPR24 receptor was accomplished by creating an T255K mutation (see, SEQ.ID.NO.:350 for nucleic acid sequence, SEQ.ID.NO.:351 for amino acid sequence), a T255K/T257R mutation (see, SEQ.ID.NO.:354 for nucleic acid sequence, SEQ.ID.NO.:355 for amino acid sequence), an 24-IC3-SST3 mutation (see, SEQ.ID.NO.:358 for nucleic acid sequence, SEQ.ID.NO.:359 for amino acid sequence), a C305Y mutation (see, SEQ.ID.NO.:362 for nucleic acid sequence, SEQ.ID.NO.:363 for amino acid sequence), a P271L mutation (see, SEQ.ID.NO.:366 for nucleic acid sequence, SEQ.ID.NO.:367 for amino acid sequence), a W269C mutation (see, SEQ.ID.NO.:370 for nucleic acid sequence, SEQ.ID.NO.:371 for amino acid sequence), a W269F mutation see, SEQ.ID.NO.:374 for nucleic acid sequence, SEQ.ID.NO.:375 for amino acid sequence), and a W269L mutation (see, SEQ.ID.NO.:378 for nucleic acid sequence, SEQ.ID.NO.:379 for amino acid sequence), a F265I mutation see, SEQ.ID.NO.:382 for nucleic acid sequence, SEQ.ID.NO.:383 for amino acid sequence), an I261Q mutation see, SEQ.ID.NO.:386 for nucleic acid sequence, SEQ.ID.NO.:387 for amino acid sequence), and a D140N mutation see, SEQ.ID.NO.:390 for nucleic acid sequence, SEQ.ID.NO.:391 for amino acid sequence).

    
    
        A. T255K Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an T255K mutation (see, SEQ.ID.NO.:350 for nucleic acid sequence, and SEQ.ID.NO.:351 for amino acid sequence). Mutagenesis was performed using Transformer Site-Directed Mutagenesis Kit (Clontech) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:348)









5′-AGAGGGTGAAACGCACAGCCATCGCCATCTG-3′






and the antisense primer (selection marker) had the following sequence:








(SEQ. ID. NO.:349)









5′-CTCCTTCGGTCCTCCTATCGTTGTCAGAAGT-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        B. T255K/T257R Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an T255K/T257R mutation (see, SEQ.ID.NO.:354 for nucleic acid sequence, and SEQ.ID.NO.:355 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:352)









5′-AGAGGGTGAAACGCAGAGCCATCGCCATCTG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:353)









5′-CAGATGGCGATGGCTCTGCGTTTCACCCTCT-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        C. 24-IC3-SST2 Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating a 24-IC3-SST2 mutation (see; SEQ.ID.NO.:358 for nucleic acid sequence, and SEQ.ID.NO.:359 for amino acid sequence). Blast result showed that GPR24 had the highest sequence homology to SST2. Thus the IC3 loop of GPR24 was replaced with that of SST2 to see if the chimera would show constitutive activity.
The BamHI-BstEII fragment containing IC3 of GPR24 was replaced with synthetic oligonucleotides that contained the IC3 of SST2. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:356)







5′-GATCCTGCAGAAGGTGAAGTCCTCTGGAATCCGAGTGGGCTCCTCTA


 


AGAGGAAGAAGTCTGAGAAGAAG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:357)







5′-GTGACCTTCTTCTCAGACTTCTTCCTCTTAGAGGAGCCCACTCGGAT


 


TCCAGAGGACTTCACCTTCTGCAG-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        D. C305Y Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an C305Y mutation (see, SEQ.ID.NO.:362 for nucleic acid sequence, and SEQ.ID.NO.:363 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:360)









5′-GGCTATGCCAACAGCTACCTCAACCCCTTTGTG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:361)









5′-CACAAAGGGGTTGAGGTAGCTGTTGGCATAGCC-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        E. P271L Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an P271L mutation (see, SEQ.ID.NO.:366 for nucleic acid sequence, and SEQ.ID.NO.:367 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:364)









5′-TTGTGTGCTGGGCACTCTACTATGTGCTACAGC-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:365)









5′-GCTGTAGCACATAGTAGAGTGCCCAGCACACAA-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        F. W269C Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an W269C mutation (see, SEQ.ID.NO.:370 for nucleic acid sequence, and SEQ.ID.NO.:371 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:368)









5′-GGTCTTCTTTGTGTGCTGCGCACCCTACTATGTG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:369)









5′-CACATAGTAGGGTGCGCAGCACACAAAGAAGACC-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        G. W269F Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an W269F mutation (see, SEQ.ID.NO.:374 for nucleic acid sequence, and SEQ.ID.NO.:375 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:372)









5′-GGTCTTCTTTGTGTGCTTCGCACCCTACTATGTG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:373)









5′-CACATAGTAGGGTGCGAAGCACACAAAGAAGACC-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        H. W269L Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an W269L mutation (see, SEQ.ID.NO.:378 for nucleic acid sequence, and SEQ.ID.NO.:379 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:376)









5′-GGTCTTCTTTGTGTGCTTGGCACCCTACTATGTG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:377)









5′-CACATAGTAGGGTGCCAAGCACACAAAGAAGACC-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        I. F265I Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an F265I mutation (see, SEQ.ID.NO.:382 for nucleic acid sequence, and SEQ.ID.NO.:383 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:380)









5′-GCCATCTGTCTGGTCATCTTTGTGTGCTGGG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:381)









5′-CCCAGCACACAAAGATGACCAGACAGATGGC-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        J. I261Q Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an I261Q mutation (see, SEQ.ID.NO.:386 for nucleic acid sequence, and SEQ.ID.NO.:387 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:384)









5′-CGCACAGCCATCGCCCAGTGTCTGGTCTTCTTTGTG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:385)









5′-CACAAAGAAGACCAGACACTGGGCGATGGCTGTGCG-3′.






The endogenous GPR24 cDNA was used as a template.

    
    
        K. D140N Mutation
    
    


Preparation of a non-endogenous version of the human GPR24 receptor was accomplished by creating an D140N mutation (see, SEQ.ID.NO.:390 for nucleic acid sequence, and SEQ.ID.NO.:391 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:388)









5′-ACCGCCATGGCCATTAACGCGTACCTGGCCACT-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:389)









5′-AGTGGCCAGGTAGCGGTTAATGGCCATGGCGGT-3′.






The endogenous GPR24 cDNA was used as a template.

3. TSHR
Preparation of non-endogenous versions of the human TSHR receptor were accomplished by creating an V509A mutation (see, SEQ.ID.NO.:394 for nucleic acid sequence, SEQ.ID.NO.:395 for amino acid sequence), a D619G mutation (see, SEQ.ID.NO.:398 for nucleic acid sequence, SEQ.ID.NO.:399 for amino acid sequence), an A623I mutation (see, SEQ.ID.NO.:402 for nucleic acid sequence, SEQ.ID.NO.:403 for amino acid sequence), a A623K mutation (see, SEQ.ID.NO.:406 for nucleic acid sequence, SEQ.ID.NO.:407 for amino acid sequence), an C672Y mutation (see, SEQ.ID.NO.:410 for nucleic acid sequence, SEQ.ID.NO.:411 for amino acid sequence), a D619G/A623K mutation (see, SEQ.ID.NO.:414 for nucleic acid sequence, SEQ.ID.NO.:415 for amino acid sequence), an V509A/C672Y mutation see, SEQ.ID.NO.:418 for nucleic acid sequence, SEQ.ID.NO.:419 for amino acid sequence), and an V509A/A623K/C672Y mutation (see, SEQ.ID.NO.:422 for nucleic acid sequence, SEQ.ID.NO.:423 for amino acid sequence).

    
    
        A. V509A Mutation
    
    


Preparation of a non-endogenous version of the human TSHR receptor was accomplished by creating an V509A mutation (see, SEQ.ID.NO.:394 for nucleic acid sequence, and SEQ.ID.NO.:395 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:392)









5′-CAAGCGAGTTATCGGCATATACGCTGACGGTC-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:393)









5′-GACCGTCAGCGTATATGCCGATAACTCGCTTG-3′.






The endogenous TSHR cDNA was used as a template. This V509A mutant can be differentiated from the endogenous version by the absence of an AccI site near the mutation site.

    
    
        B. D619G Mutation
    
    


Preparation of a non-endogenous version of the human TSHR receptor was also accomplished by creating a D619G mutation (see, SEQ.ID.NO.:398 for nucleic acid sequence, and SEQ.ID.NO.:399 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:396)









5′-ACCCAGGGGACAAAGGTACCAAAATTGCCAA-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:397)









5′-TTGGCAATTTTGGTACCTTTGTCCCCTGGGT-3′.






The endogenous TSHR cDNA was used as a template. This D619G mutant can be differentiated from the endogenous version by the presence of a KpnI site near the mutation site.

    
    
        C. A623I Mutation
    
    


Preparation of a non-endogenous version of the human TSHR was accomplished by creating an A623I mutation (see, SEQ.ID.NO.:402 for nucleic acid sequence, and SEQ.ID.NO.:403 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:400)









5′-AAAGATACCAAAATTATCAAGAGGATGGCTGT-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:401)









5′-ACAGCCATCCTCTTGATAATTTTGGTATCTTT-3′.






The endogenous TSHR cDNA was used as a template. This A623I mutant can be differentiated from the endogenous version by the absence of a BstXI site near the mutation site.

    
    
        D. A623K Mutation
    
    


Preparation of a non-endogenous version of the human TSHR receptor was also accomplished by creating a A623K mutation (see, SEQ.ID.NO.:406 for nucleic acid sequence, and SEQ.ID.NO.:407 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:404)









5′-AAAGATACCAAAATTAAGAAGAGGATGGCTGTG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:405)









5′-CACAGCCATCCTCTTCTTAATTTTGGTATCTTT-3′.






The endogenous TSHR cDNA was used as a template. This A623K mutant can be differentiated from the endogenous version by the absence of a BstXI site near the mutation site.

    
    
        E. C672Y Mutation
    
    


Preparation of a non-endogenous version of the human TSHR receptor was also accomplished by creating a C672Y mutation (see, SEQ.ID.NO.:410 for nucleic acid sequence, and SEQ.ID.NO.:411 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:408)









5′-CTATCCACTTAACTCGTACGCCAATCCATTCCTC-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:409)









5′-GAGGAATGGATTGGCGTACGAGTTAAGTGGATAG-3′.






The endogenous TSHR cDNA was used as a template. This C672Y mutant can be differentiated from the endogenous version by the presence of a BsiWI site near the mutation site.

    
    
        F. D619G/A623K Mutation
    
    


Preparation of a non-endogenous version of the human TSHR receptor was also accomplished by creating a D619G/A623K mutation (see, SEQ.ID.NO.:414 for nucleic acid sequence, and SEQ.ID.NO.:415 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The PCR sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:412)







5′-ACCCAGGGGACAAAGGTACCAAAATTAAGAAGAGGATGGCTGTG-3′






and the antisense primer had the following sequence:








(SEQ. ID. NO.:413)









5′-CACAGCCATCCTCTTCTTAATTTTGGTACCTTTGTCCCCT



 



GGGT-3′.






The non-endogenous D619G mutant version of TSHR cDNA was used as a template. This D619G/A623K mutant can be differentiated from the endogenous version by the presence of a KpnI site near the D619G mutation site and absence of a BstXI site near the A623K mutation site.

    
    
        G. V509A/C672Y Mutation
    
    


Preparation of a non-endogenous version of the human TSHR receptor was also accomplished by creating a V509A/C672Y mutation (see, SEQ.ID.NO.:418 for nucleic acid sequence, and SEQ.ID.NO.:419 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The V509A sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:416)









5′-CAAGCGAGTTATCGGCATATACGCTGACGGTC-3′






and the C672Y antisense primer had the following sequence:








(SEQ. ID. NO.:417)









5′-GAGGAATGGATTGGCGTACGAGTTAAGTGGATAG-3′.






The endogenous TSHR cDNA was used as a template. This V509A/C672Y mutant can be differentiated from the endogenous version by the absence of an AccI site near the V509A mutation site and presence of a BsiWI site near the C672Y mutation site.

    
    
        H. V509A/A623K/C672Y Mutation
    
    


Preparation of a non-endogenous version of the human TSHR receptor was also accomplished by creating a V509A/A623K/C672Y mutation (see, SEQ.ID.NO.:422 for nucleic acid sequence, and SEQ.ID.NO.:423 for amino acid sequence). Mutagenesis was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer. The A623K sense mutagenesis primer used had the following sequence:







(SEQ. ID. NO.:420)









5′-AAAGATACCAAAATTAAGAAGAGGATGGCTGTG-3′






and the A623K antisense primer had the following sequence:








(SEQ. ID. NO.:421)









5′-CACAGCCATCCTCTTCTTAATTTTGGTATCTTT-3′.






The non-endogenous V509A/C672Y mutant version of TSHR cDNA was used as a template. This V509A/A623K/C672Y mutant can be differentiated from the endogenous version by the absence of an AccI site near the V509A mutation site, absence of a BstXI near the A623K mutation site and the presence of a BsiWI site near the C672Y mutation site.

The non-endogenous human GPCRs were then sequenced and the derived and verified nucleic acid and amino acid sequences are listed in the accompanying “Sequence Listing” appendix to this patent document, as summarized in Table G below:









TABLE G






Nucleic Acid Sequence
Amino Acid Sequence


Mutated GPCR
Listing
Listing







5HT-1A
SEQ.ID.NO.: 424
SEQ.ID.NO.: 425


V343K


5HT-1B
SEQ.ID.NO.: 426
SEQ.ID.NO.: 427


T313K


5HT-1D
SEQ.ID.NO.: 428
SEQ.ID.NO.: 429


T300K


5HT-1E
SEQ.ID.NO.: 430
SEQ.ID.NO.: 431


A290K


5HT-1F
SEQ.ID.NO.: 432
SEQ.ID.NO.: 433


A292K


5HT-2B
SEQ.ID.NO.: 434
SEQ.ID.NO.: 435


S323K


5HT-4A
SEQ.ID.NO.: 436
SEQ.ID.NO.: 437


A258K


5HT-4B
SEQ.ID.NO.: 438
SEQ.ID.NO.: 439


A258K


5HT-4C
SEQ.ID.NO.: 440
SEQ.ID.NO.: 441


A258K


5HT-4D
SEQ.ID.NO.: 442
SEQ.ID.NO.: 443


A258K


5HT-4E
SEQ.ID.NO.: 444
SEQ.ID.NO.: 445


A258K


5HT-5A
SEQ.ID.NO.: 446
SEQ.ID.NO.: 447


A284K


5HT-6
SEQ.ID.NO.: 448
SEQ.ID.NO.: 449


S267K


5HT-7
SEQ.ID.NO.: 450
SEQ.ID.NO.: 451


A326K


AVPR1A
SEQ.ID.NO.: 452
SEQ.ID.NO.: 453


V290K


AVPR1B
SEQ.ID.NO.: 454
SEQ.ID.NO.: 455


V280K


AVPR2
SEQ.ID.NO.: 456
SEQ.ID.NO.: 457


V270K


BBR3
SEQ.ID.NO.: 458
SEQ.ID.NO.: 459


A270K


BDKR1
SEQ.ID.NO.: 460
SEQ.ID.NO.: 461


T249K


BDKR2
SEQ.ID.NO.: 462
SEQ.ID.NO.: 463


T269K


C3a
SEQ.ID.NO.: 464
SEQ.ID.NO.: 465


F376K


C5a
SEQ.ID.NO.: 466
SEQ.ID.NO.: 467


L241K


CB1
SEQ.ID.NO.: 468
SEQ.ID.NO.: 469


A342K


CB2
SEQ.ID.NO.: 470
SEQ.ID.NO.: 471


A244K


CCR2b
SEQ.ID.NO.: 472
SEQ.ID.NO.: 473


V242K


CCR3
SEQ.ID.NO.: 474
SEQ.ID.NO.: 475


I238K


CCR5
SEQ.ID.NO.: 476
SEQ.ID.NO.: 477


V234K


CCR8
SEQ.ID.NO.: 478
SEQ.ID.NO.: 479


I237K


CCR9
SEQ.ID.NO.: 480
SEQ.ID.NO.: 481


L253K


CRFR1
SEQ.ID.NO.: 482
SEQ.ID.NO.: 483


T316P


CXCR4
SEQ.ID.NO.: 484
SEQ.ID.NO.: 485


L238K


Dopamine D1
SEQ.ID.NO.: 486
SEQ.ID.NO.: 487


L271K


Dopamine D2
SEQ.ID.NO.: 488
SEQ.ID.NO.: 489


T372K


Dopamine D3
SEQ.ID.NO.: 490
SEQ.ID.NO.: 491


T328K


Dopamine D5
SEQ.ID.NO.: 492
SEQ.ID.NO.: 493


L295K


ETA
SEQ.ID.NO.: 494
SEQ.ID.NO.: 495


A305K


ETB
SEQ.ID.NO.: 496
SEQ.ID.NO.: 497


A322K


FPR1
SEQ.ID.NO.: 498
SEQ.ID.NO.: 499


L240K


FPRL1
SEQ.ID.NO.: 500
SEQ.ID.NO.: 501


L240K


GALR1
SEQ.ID.NO.: 502
SEQ.ID.NO.: 503


A246K


GALR2
SEQ.ID.NO.: 504
SEQ.ID.NO.: 505


T235K


GIP
SEQ.ID.NO.: 506
SEQ.ID.NO.: 507


T343P


mGluR1
SEQ.ID.NO.: 346
SEQ.ID.NO.: 347


3′ Deletion


GPR5
SEQ.ID.NO.: 508
SEQ.ID.NO.: 509


V224K


GPR24 (also known as


MCH or SLC-1)


T255K
SEQ.ID.NO.: 350
SEQ.ID.NO.: 351


T255K/T257R
SEQ.ID.NO.: 354
SEQ.ID.NO.: 355


24-IC3-SST2
SEQ.ID.NO.: 358
SEQ.ID.NO.: 359


C305Y
SEQ.ID.NO.: 362
SEQ.ID.NO.: 363


P271L
SEQ.ID.NO.: 366
SEQ.ID.NO.: 367


W269C
SEQ.ID.NO.: 370
SEQ.ID.NO.: 371


W269F
SEQ.ID.NO.: 374
SEQ.ID.NO.: 375


W269L
SEQ.ID.NO.: 378
SEQ.ID.NO.: 379


F265I
SEQ.ID.NO.: 382
SEQ.ID.NO.: 383


I261Q
SEQ.ID.NO.: 386
SEQ.ID.NO.: 387


D140N
SEQ.ID.NO.: 390
SEQ.ID.NO.: 391


GRPR
SEQ.ID.NO.: 510
SEQ.ID.NO.: 511


A263K


M1
SEQ.ID.NO.: 512
SEQ.ID.NO.: 513


A364K


M2
SEQ.ID.NO.: 514
SEQ.ID.NO.: 515


T386K


M3
SEQ.ID.NO.: 516
SEQ.ID.NO.: 517


A490K


M4
SEQ.ID.NO.: 518
SEQ.ID.NO.: 519


T399K


M5
SEQ.ID.NO.: 520
SEQ.ID.NO.: 521


A441K


MC3
SEQ.ID.NO.: 522
SEQ.ID.NO.: 523


A241K


NK1R
SEQ.ID.NO.: 524
SEQ.ID.NO.: 525


V247K


NK2R
SEQ.ID.NO.: 526
SEQ.ID.NO.: 527


V249K


NK3R
SEQ.ID.NO.: 528
SEQ.ID.NO.: 529


V298K


NMBR
SEQ.ID.NO.: 530
SEQ.ID.NO.: 531


A265K


NPY5
SEQ.ID.NO.: 532
SEQ.ID.NO.: 533


A297K


NTSR1
SEQ.ID.NO.: 534
SEQ.ID.NO.: 535


V302K


NTSR2
SEQ.ID.NO.: 536
SEQ.ID.NO.: 537


V269K


OPRD
SEQ.ID.NO.: 538
SEQ.ID.NO.: 539


T260K


OPRL1
SEQ.ID.NO.: 540
SEQ.ID.NO.: 541


T262K


OPRK
SEQ.ID.NO.: 542
SEQ.ID.NO.: 543


T273K


OPRM
SEQ.ID.NO.: 544
SEQ.ID.NO.: 545


T281K


OPRM1A
SEQ.ID.NO.: 546
SEQ.ID.NO.: 547


T281K


OX1R
SEQ.ID.NO.: 548
SEQ.ID.NO.: 549


F367K


OX2R
SEQ.ID.NO.: 550
SEQ.ID.NO.: 551


A297K


PACAP
SEQ.ID.NO.: 552
SEQ.ID.NO.: 553


T355K


PAF
SEQ.ID.NO.: 554
SEQ.ID.NO.: 555


L231K


PGE EP1
SEQ.ID.NO.: 556
SEQ.ID.NO.: 557


V296K


PGE EP2
SEQ.ID.NO.: 558
SEQ.ID.NO.: 559


L263K


PGE EP4
SEQ.ID.NO.: 560
SEQ.ID.NO.: 561


V271K


PTHR1
SEQ.ID.NO.: 562
SEQ.ID.NO.: 563


T410P


PTHR2
SEQ.ID.NO.: 564
SEQ.ID.NO.: 565


T365P


SCTR
SEQ.ID.NO.: 566
SEQ.ID.NO.: 567


T344P


SST1
SEQ.ID.NO.: 568
SEQ.ID.NO.: 569


T290K


SST2
SEQ.ID.NO.: 570
SEQ.ID.NO.: 571


T255K


SST3
SEQ.ID.NO.: 572
SEQ.ID.NO.: 573


T256K


SST4
SEQ.ID.NO.: 574
SEQ.ID.NO.: 575


T258K


SST5
SEQ.ID.NO.: 576
SEQ.ID.NO.: 577


T247K


TSHR


V509A
SEQ.ID.NO.: 394
SEQ.ID.NO.: 395


D619G
SEQ.ID.NO.: 398
SEQ.ID.NO.: 399


A623I
SEQ.ID.NO.: 402
SEQ.ID.NO.: 403


A623K
SEQ.ID.NO.: 406
SEQ.ID.NO.: 407


C672Y
SEQ.ID.NO.: 410
SEQ.ID.NO.: 411


D619G/A623K
SEQ.ID.NO.: 414
SEQ.ID.NO.: 415


V509A/C672Y
SEQ.ID.NO.: 418
SEQ.ID.NO.: 419


V509A/A623K/C672Y
SEQ.ID.NO.: 422
SEQ.ID.NO.: 423


VIPR
SEQ.ID.NO.: 578
SEQ.ID.NO.: 579


T343P


VIPR2
SEQ.ID.NO.: 580
SEQ.ID.NO.: 581


T330P









Assessment of constitutive activity of the non-endogenous versions of the known GPCRs can then be accomplished.
Example 3
Receptor Expression
Although a variety of cells are available to the art for the expression of proteins, it is most preferred that mammalian cells be utilized. The primary reason for this is predicated upon practicalities, i.e., utilization of, e.g., yeast cells for the expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor-coupling, genetic-mechanism and secretary pathways that have evolved for mammalian systems—thus, results obtained in non-mammalian cells, while of potential use, are not as preferred as that obtained from mammalian cells. Of the mammalian cells, COS-7, Hek-293 and Hek-293T cells are particularly preferred, although the specific mammalian cell utilized can be predicated upon the particular needs of the artisan. The following approach was used for the indicated receptors, and can also be applied with respect to other receptors disclosed herein.
On day one, 2×104 Hek-293T cells well were plated out. On day two, two reaction tubes were prepared (the proportions to follow for each tube are per plate): tube A was prepared by mixing 20 μg DNA (e.g., pCMV vector; pCMV vector with receptor cDNA, etc.) in 1.2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); tube B was prepared by mixing 120 μl lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B were admixed by inversions (several times), followed by incubation at room temperature for 30-45 min. The admixture is referred to as the “transfection mixture”. Plated Hek-293T cells were washed with 1×PBS, followed by addition of 10 ml serum free DMEM. 2.4 ml of the transfection mixture were added to the cells, followed by incubation for 4 hrs at 37° C./5% CO2. The transfection mixture was removed by aspiration, followed by the addition of 25 ml of DMEM/10% Fetal Bovine Serum. Cells were incubated at 37° C./5% CO2. After 72 hr incubation, cells were harvested and utilized for analysis.
Example 4
Assays for Determination of Constitutive Activity of Non-Endogenous GPCRs
A variety of approaches are available for assessment of constitutive activity of the non-endogenous versions of known GPCRs. The following are illustrative; those of ordinary skill in the art are credited with the ability to determine those techniques that are preferentially beneficial for the needs of the artisan.
1. Membrane Binding Assays: [35S]GTPγS Assay
When a G protein-coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Constitutively activated receptors continue to exchange GDP for GTP. The non-hydrolyzable GTP analog, [35S]GTPγS, can be utilized to demonstrate enhanced binding of [35S]GTPγS to membranes expressing constitutively activated receptors. The advantage of using [35S]GTPγS binding to measure constitutive activation is that: (a) it is generically applicable to all G protein-coupled receptors; (b) it is proximal at the membrane surface making it less likely to pick-up molecules which affect the intracellular cascade.
The assay utilizes the ability of G protein coupled receptors to stimulate [35S]GTPγS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to known, and constitutively activated G protein-coupled receptors. The assay is generic and has application to drug discovery at all G protein-coupled receptors.
The [35S]GTPγS assay can be incubated in 20 mM HEPES and between 1 and about 20 mM MgCl2 (this amount can be adjusted for optimization of results, although 20 mM is preferred) pH 7.4, binding buffer with between about 0.3 and about 1.2 nM [35S]GTPγS (this amount can be adjusted for optimization of results, although 1.2 is preferred) and 12.5 to 75 μg membrane protein (e.g, COS-7 cells expressing the receptor; this amount can be adjusted for optimization, although 75 μg is preferred) and 1 μM GDP (this amount can be changed for optimization) for 1 hour. Wheatgerm agglutinin beads (25 μl; Amersham) should then be added and the mixture incubated for another 30 minutes at room temperature. The tubes are then centrifuged at 1500×g for 5 minutes at room temperature and then counted in a scintillation counter.
A less costly but equally applicable alternative has been identified which also meets the needs of large scale screening. FLASH PLATESTM and WALLACTM scintistrips may be utilized to format a high throughput [35S]GTPγS binding assay. Furthermore, using this technique, the assay can be utilized for known GPCRs to simultaneously monitor tritiated ligand binding to the receptor at the same time as monitoring the efficacy via [35S]GTPγS binding. This is possible because the Wallac beta counter can switch energy windows to look at both tritium and 35S-labeled probes. This assay may also be used to detect other types of membrane activation events resulting in receptor activation. For example, the assay may be used to monitor 32P phosphorylation of a variety of receptors (both G protein coupled and tyrosine kinase receptors). When the membranes are centrifuged to the bottom of the well, the bound [35S]GTPγS or the 32P-phosphorylated receptor will activate the scintillant which is coated of the wells. SCINTI® strips (Wallac) have been used to demonstrate this principle. In addition, the assay also has utility for measuring ligand binding to receptors using radioactively labeled ligands. In a similar manner, when the radiolabeled bound ligand is centrifuged to the bottom of the well, the scintistrip label comes into proximity with the radiolabeled ligand resulting in activation and detection.
2. Membrane-Based cAMP
A FLASH PLATETM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes. The Flash Plate wells contain a scintillant coating which also contains a specific antibody recognizing cAMP. The cAMP generated in the wells was quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in membranes that express the receptors.
Transfected cells are harvested approximately three days after transfection. Membranes were prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl2. Homogenization is performed on ice using a Brinkman POLYTRONTM for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000×g for 15 minutes at 4° C. The resulting pellet is then resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000×g for 15 minutes at 4° C. The resulting pellet can be stored at −80° C. until utilized. On the day of measurement, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCL2 (these amounts can be optimized, although the values listed herein are preferred), to yield a final protein concentration of 0.60 mg/ml (the resuspended membranes were placed on ice until use).
cAMP standards and Detection Buffer (comprising 2 μCi of tracer [125I cAMP (100 μl)] to 11 ml Detection Buffer) are prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer is prepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mM MgCl2, 20 mM (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer can be stored on ice until utilized. The assay is initiated by the addition of 50 μL of assay buffer followed by addition of 50 μL of membrane suspension to the NEN Flash Plate. The resultant assay mixture is incubated for 60 minutes at room temperature followed by addition of 100 μL of detection buffer. Plates are then incubated an additional 2-4 hours followed by counting in a Wallac MICROBETATM scintillation counter. Values of cAMP/well are extrapolated from a standard cAMP curve that is contained within each assay plate.
3. Cell-Based cAMP for Gi Coupled Target GPCRs
TSHR is a Gs coupled GPCR that causes the accumulation of cAMP upon activation. TSHR was constitutively activated by mutating amino acid residue 623 (i.e., changing an alanine residue to an isoleucine residue). See, SEQ.ID.NO.:402 for nucleic acid sequence and SEQ.ID.NO.:403 for deduced amino acid sequence. A Gi coupled receptor is expected to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging. An effective technique for measuring the decrease in production of cAMP as an indication of constitutive activation of a Gi coupled receptor can be accomplished by co-transfecting, most preferably, non-endogenous, constitutively activated TSHR (TSHR-A623I) (or an endogenous, constitutively active Gs coupled receptor) as a “signal enhancer” with a Gi linked target GPCR, such as GPR24, to establish a baseline level of cAMP. Upon creating a non-endogenous version of the Gi coupled receptor, this non-endogenous version of the target GPCR is then co-transfected with the signal enhancer, and it is this material that can be used for screening. We utilized such approach to effectively generate a signal when a cAMP assay is used; this approach is preferably used in the direct identification of candidate compounds against Gi coupled receptors. It is noted that for a Gi coupled GPCR, when this approach is used, an inverse agonist of the target GPCR will increase the cAMP signal and an agonist will decrease the cAMP signal.
On day one, 2×104 Hek-293 and Hek-293T cells/well were plated out. On day two, two reaction tubes were prepared (the proportions to follow for each tube are per plate): tube A was prepared by mixing 2 μg DNA of each receptor transfected into the mammalian cells, for a total of 4 μg DNA (e.g., pCMV vector; pCMV vector with mutated THSR (TSHR-A623I); TSHR-A623I and GPR24, etc.) in 1.2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); tube B was prepared by mixing 120 μl lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B were then admixed by inversion (several times), followed by incubation at room temperature for 30-45 min. The admixture is referred to as the “transfection mixture”. Plated Hek-293 cells were washed with 1×PBS, followed by addition of 10 ml serum free DMEM. 2.4 ml of the transfection mixture was then added to the cells, followed by incubation for 4 hrs at 37° C./5% CO2. The transfection mixture was then removed by aspiration, followed by the addition of 25 ml of DMEM/10% Fetal Bovine Serum. Cells were then incubated at 37° C./5% CO2. After 24 hr incubation, cells were then harvested and utilized for analysis.
A FLASH PLATETM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes. The Flash Plate wells can contain a scintillant coating which also contains a specific antibody recognizing cAMP. The cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express the receptors.
Transfected cells were harvested approximately twenty four hours after transient transfection. Media was carefully aspirated and discarded. Ten milliliters of PBS was gently added to each dish of cells followed by careful aspiration. One milliliter of Sigma cell dissociation buffer and 3 ml of PBS are added to each plate. Cells were pipetted off the plate and the cell suspension is collected into a 50 ml conical centrifuge tube. Cells were then centrifuged at room temperature at 1,100 rpm for 5 min. The cell pellet was carefully re-suspended into an appropriate volume of PBS (about 3 ml/plate). The cells were then counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final concentration of about 50 μl/well).
cAMP standards and Detection Buffer (comprising 1 μCi of tracer [125I cAMP (50 μl] to 11 ml Detection Buffer) was prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer should be prepared fresh for screening and contained 50 μL of Stimulation Buffer, 3 μL of test compound (12 μM final assay concentration) and 50 μL cells, Assay Buffer can be stored on ice until utilized. The assay can be initiated by addition of 50 μL of cAMP standards to appropriate wells followed by addition of 50 μL of PBSA to wells H-11 and H12. Fifty μL of Stimulation Buffer was added to all wells. Selected compounds (e.g., TSH, 100 nM MCH, MCH/TSH) were added to appropriate wells using a pin tool capable of dispensing 3 μL of compound solution, with a final assay concentration of 12 μM test compound and 100 μL total assay volume. The cells were then added to the wells and incubated for 60 min at room temperature. 100 μL of Detection Mix containing tracer cAMP was then added to the wells. Plates were then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well were then extrapolated from a standard cAMP curve which is contained within each assay plate.
FIG. 1 evidences about a 22% decrease in cAMP production of cells co-transfected with TSHR-A623I (in the presence of TSH) and non-endogenous, constitutively activated GPR24 (“24-IC3-SST2”) (262.266 pmol cAMP/well) compared to TSHR-A623I with endogenous GPR24 (“GPR24 wt”) (336.50293 pmol cAMP/well). Co-transfection of TSHR-A623I with non-endogenous, constitutively activated GPR24 (“I261Q”) evidences about a 27% decrease in production of cAMP when compared to “GPR24 wt.” Such a decrease in cAMP production signifies that non-endogenous version of GPR24 (“I261Q”) is constitutively active. Thus, a candidate compound which impacts the GPR24 receptor by increasing the cAMP signal is an inverse agonist, while a GPR24 agonist will decrease the cAMP signal. Based upon the data generated for FIG. 1, 24-IC3-SST2 and I261Q are most preferred non-endogenous versions of GPR24 when used in a TSHR (constitutively activated co-transfection approach using a cAMP assay.
FIG. 2 evidences about a 60% decrease in cAMP production of cells co-transfected with TSHR-A623I (in the presence of TSH) and non-endogenous, constitutively activated GPR5 (“V224K”) (23.5 pmole cAMP/well) compared to TSHR-A623I with endogenous GPR5 (“GPR5 wt”) (58.79 pmol cAMP/well). About a 78% and about a 45% decrease in production of cAMP was evidenced when comparing TSHR-A623I co-transfected with “V225K” and TSHR-A623I co-transfected with “GPR5 wt” against pCMV co-transfected with TSHR-A623I (106.75 pmol cAMP/well), respectively. As mentioned above, a decrease in cAMP production evidences a constitutively active GPR5. Thus, a preferred candidate compound (i.e., an inverse agonist) would likely bind the Gi coupled receptor to increase the signal of activation.
Preferably, and as noted previously, to ensure that a small molecule candidate compound is targeting the Gi coupled target receptor and not, for example, the TSHR-A623I, the directly identified candidate compound is preferably screened against the signal enhancer in the absence of the target receptor.
C. Reporter-Based Assays

    
    
        1. CRE-Luc Reporter Assay (Gs-Associated Receptors)
    
    


A method to detect Gs stimulation depends on the known property of the transcription factor CREB, which is activated in a cAMP-dependent manner. A PATHDETECTTM CREB trans-Reporting System (Stratagene, Catalogue #219010) can utilized to assay for Gs coupled activity in 293 or 293T cells. Cells are transfected with the plasmids components of this above system and the indicated expression plasmid encoding endogenous or mutant receptor using a Mammalian Transfection Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions. Briefly, 400 ng pFR-Luc (luciferase reporter plasmid containing Gal4 recognition sequences), 40 ng pFA2-CREB (Gal4-CREB fusion protein containing the Gal4 DNA-binding domain), 80 ng pCMV-receptor expression plasmid (comprising the receptor) and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples) are combined in a calcium phosphate precipitate per the Kit's instructions. Half of the precipitate is equally distributed over 3 wells in a 96-well plate, kept on the cells overnight, and replaced with fresh medium the following morning. Forty-eight (48) hr after the start of the transfection, cells are treated and assayed for, e.g., luciferase activity.

    
    
        2. 8×CRE-Luc Reporter Assay
    
    


HEK-293T cells are plated-out on 96 well plates at a density of 3×104 cells per well and were transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions. A DNA/lipid mixture is prepared for each 6-well transfection as follows: 260 ng of plasmid DNA in 100 μl of DMEM were gently mixed with 2 μl of lipid in 100 μl of DMEM (the 260 ng of plasmid DNA consisted of 200 ng of a 8×CRE-Luc reporter plasmid (see below and FIG. 1 for a representation of a portion of the plasmid), 50 ng of pCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone, and 10 ng of a GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)). The 8×CRE-Luc reporter plasmid was prepared as follows: vector SRIF-β-gal was obtained by cloning the rat somatostatin promoter (−71/+51) at BglV-HindIII site in the pβgal-Basic Vector (Clontech). Eight (8) copies of cAMP response element were obtained by PCR from an adenovirus template AdpCF126CCRE8 (see, 7 Human Gene Therapy 1883 (1996)) and cloned into the SRIF-β-gal vector at the Kpn-BglV site, resulting in the 8×CRE-β-gal reporter vector. The 8×CRE-Luc reporter plasmid was generated by replacing the beta-galactosidase gene in the 8×CRE-β-gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the HindIII-BamHI site. Following 30 min. incubation at room temperature, the DNA/lipid mixture was diluted with 400 μl of DMEM and 100 μl of the diluted mixture was added to each well. 100 μl of DMEM with 10% FCS were added to each well after a 4 hr incubation in a cell culture incubator. The following day the transfected cells were changed with 200 μl/well of DMEM with 10% FCS. Eight (8) hours later, the wells were changed to 100 μl/well of DMEM without phenol red, after one wash with PBS. Luciferase activity were measured the next day using the LucLiteTM reporter gene assay kit (Packard) following manufacturer instructions and read on a 1450 MicroBetaTM scintillation and luminescence counter (Wallac).
FIG. 3A represents about a 63% increase in activity of the non-endogenous, constitutively active version of human Dopamine D1 receptor (189270 relative light units) compared with that of the endogenous Dopamine D1 (70622 relative light units).
FIG. 3B represents about a 48% decreases in activity of the non-endogenous, constitutively active version of human OPRM (a Gi coupled receptor; see Example 4(3)), about a 53% decrease in activity of the non-endogenous, constitutively active version of human 5-HT1A (a Gi coupled receptor; see Example 4(3)), about a 91% increase in activity of the non-endogenous, constitutively active version of human 5-HT1B, and about a 20% increase in activity of the non-endogenous, constitutively active version of human 5-HT2B over the respective endogenous version of the GPCR.
FIG. 3C represents about a 29% increase in activity of the non-endogenous, constitutively active version of human CCR3, about a 41% increase in activity of the non-endogenous, constitutively active version of human NTSR1, about a 51% increase in activity of the non-endogenous, constitutively active version of human CB2, and about a 40% decrease in activity of the non-endogenous, constitutively active version of human CXCR4 (a Gi coupled receptor; see Example 4(3)) over the respective endogenous version of the GPCR.
FIG. 3D represents about a 75% increase in activity of the non-endogenous, constitutively active version of human PTHR1, about a 74% increase in activity of the non-endogenous, constitutively active version of human PTHR2, about a 56% increase in activity of the non-endogenous, constitutively active version of human SCTR, about a 96% increase in activity of the non-endogenous, constitutively active version of human PACAP, about a 88% increase in activity of the non-endogenous, constitutively active version of human VIPR1, and about a 91% increase in activity of the non-endogenous, constitutively active version of human VIPR2 over the respective endogenous version of the GPCR.
FIG. 3E represents about a 51% increase in activity of the non-endogenous, constitutively active version of human NTSR1, about a 31% decrease in activity of the non-endogenous, constitutively active version of human M1, about a 19% decrease in activity of the non-endogenous, constitutively active version of human M2, about a 32% increase in activity of the non-endogenous, constitutively active version of human M3, about a 33% decrease in activity of the non-endogenous, constitutively active version of human M4, about a 17% decrease in activity of the non-endogenous, constitutively active version of human M5, and about a 60% increase in activity of the non-endogenous, constitutively active version of human 5-HT1D over the respective endogenous version of the GPCR. M2, M4 and 5-HT1D are indicated as being Gi coupled while NTSR1, M1, M3 and M5 are indicated as being Gq coupled.

    
    
        3. AP1 Reporter Assay (Gq-Associated Receptors)
    
    


A method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing AP1 elements in their promoter. A PATHDETECTTM AP-1 cis-Reporting System (Stratagene, Catalog #219073) can be utilized following the protocol set forth above with respect to the CREB reporter assay, except that the components of the calcium phosphate precipitate were 410 ng pAP1-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.

    
    
        4. SRF-Luc Reporter Assay (Gq-Associated Receptors)
    
    


One method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing serum response factors in their promoter. A PATHDETECTTM SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gq coupled activity in, e.g., COS7 cells. Cells are transfected with the plasmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a MAMMALIAN TRANSFECTIONTM Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples) are combined in a calcium phosphate precipitate as per the manufacturer's instructions. Half of the precipitate is equally distributed over 3 wells in a 96-well plate, kept on the cells in a serum free media for 24 hours. The last 5 hours the cells are incubated with 1 mM Angiotensin, where indicated. Cells are then lysed and assayed for luciferase activity using a LUCLITETM Kit (Packard, Cat. #6016911) and “Trilux 1450 Microbeta” liquid scintillation and luminescence counter (Wallac) per the manufacturer's instructions. The data can be analyzed using GRAPHPAD PRISMTM 2.0a (GraphPad Software Inc.).

    
    
        5. Intracellular IP3 Accumulation Assay (Gq-Associated Receptors)
    
    


On day 1, cells comprising the receptors (endogenous and/or non-endogenous) can be plated onto 24 well plates, usually 1×105 cells/well (although this number can be optimized. On day 2 cells can be transfected by firstly mixing 0.25 μg DNA in 50 μL serum free DMEM/well and 2 μL lipofectamine in 50 μl serum-free DMEM/well. The solutions are gently mixed and incubated for 15-30 min at room temperature. Cells are washed with 0.5 ml PBS and 400 μl of serum free media is mixed with the transfection media and added to the cells. The cells are then incubated for 3-4 hrs at 37° C./5% CO2 and then the transfection media is removed and replaced with 1 ml/well of regular growth media. On day 3 the cells are labeled with 3H-myo-inositol. Briefly, the media is removed and the cells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum free media (GIBCO BRL) is added/well with 0.25 μCi of 3H-myo-inositol/well and the cells are incubated for 16-18 hrs o/n at 37° C./5% CO2. On Day 4 the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium is added containing inositol-free/serum free media 10 μM pargyline 10 mM lithium chloride or 0.4 ml of assay medium and 50 μL of 10× ketanserin (ket) to final concentration of 10 μM. The cells are then incubated for 30 min at 37° C. The cells are then washed with 0.5 ml PBS and 200 μL of fresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added/well. The solution is kept on ice for 5-10 min or until cells were lysed and then neutralized by 200 μl of fresh/ice cold neutralization sol. (7.5% HCL). The lysate is then transferred into 1.5 ml eppendorf tubes and 1 ml of chloroform/methanol (1:2) is added/tube. The solution is vortexed for 15 sec and the upper phase is applied to a Biorad AG1-X8TM anion exchange resin (100-200 mesh). Firstly, the resin is washed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded onto the column. The column is washed with 10 mls of 5 mM myo-inositol and 10 ml of 5 mM Na-borate/60 mM Na-formate. The inositol tris phosphates are eluted into scintillation vials containing 10 ml of scintillation cocktail with 2 ml of 0.1 M formic acid/1 M ammonium formate. The columns are regenerated by washing with 10 ml of 0.1 M formic acid/3M ammonium formate and rinsed twice with H2O and stored at 4° C. in water.
FIG. 4 represents two preferred non-endogenous, constitutively activated exemplary versions of GPR24, 24-IC3-SST2 and I261Q, for use in an IP3 assay. When compared to the endogenous version of GPR24 (“GPR24 wt”), 24-IC3-SST2 evidenced about a 27% increase in IP3 accumulation, while the I26Q version represented and about a 32% increase.
Example 5
GPCR Fusion Protein Preparation
The design of the constitutively activated GPCR-G protein fusion construct was accomplished as follows: both the 5′ and 3′ ends of the rat G protein Gsα (long form; Itoh, H. et al., 83 PNAS 3776 (1986)) were engineered to include a HindIII (5′-AAGCTT-3′) sequence thereon. Following confirmation of the correct sequence (including the flanking HindIII sequences), the entire sequence was shuttled into pcDNA3.1(−) (Invitrogen, cat. no. V795-20) by subcloning using the HindIII restriction site of that vector. The correct orientation for the Gsα sequence was determined after subcloning into pcDNA3.1(−). The modified pcDNA3.1(−) containing the rat Gsα gene at HindIII sequence was then verified; this vector was now available as a “universal” Gsα protein vector. The pcDNA3.1(−) vector contains a variety of well-known restriction sites upstream of the HindIII site, thus beneficially providing the ability to insert, upstream of the Gs protein, the coding sequence of an endogenous, constitutively active GPCR. This same approach can be utilized to create other “universal” G protein vectors, and, of course, other commercially available or proprietary vectors known to the artisan can be utilized—the important criteria is that the sequence for the GPCR be upstream and in-frame with that of the G protein.
1. TSHR-Gsα Fusion Protein

    
    
        a. Stable Cell Line Production for TSHR
    
    


Approximately 1.2 to 1.3×107 HEK-293 cells are plated on a 15 cm tissue culture plate. Grown in DME High Glucose Medium containing ten percent fetal bovine serum and one percent sodium pyruvate, L-glutamine, and antibiotics. Twenty-four hours following plating of 293 cells to  ̃80% confluency, the cells are transfected using 12 μg of DNA. The 12 μg of DNA is combined with 60 μL of lipofectamine and 2 mL of DME High Glucose Medium without serum. The medium is aspirated from the plates and the cells are washed once with medium without serum. The DNA, lipofectamine, and medium mixture is added to the plate along with 10 mL of medium without serum. Following incubation at 37° C. for four to five hours, the medium is aspirated and 25 ml of medium containing serum is added. Twenty-four hours following transfection, the medium is aspirated again, and fresh medium with serum is added. Forty-eight hours following transfection, the medium is aspirated and medium with serum is added containing geneticin (G418 drug) at a final concentration of 500 μg/mL. The transfected cells now undergo selection for positively transfected cells containing the G418 resistant gene. The medium is replaced every four to five days as selection occurs. During selection, cells are grown to create stable pools, or split for stable clonal selection.

    
    
        b. TSHR(A623K) Fusion Protein
    
    


TSHR-Gsα Fusion Protein construct was then made as follows: primers were designed for both endogenous, constitutively activated and non-endogenous, constitutively activated TSHR were as follows:









(SEQ. ID. NO.:582; sense)



5′-gatc[TCTAGA]ATGAGGCCGGCGGACTTGCTGC-3′



 



(SEQ. ID. NO.:583; antisense)



5′-ctag[GATATC]CGCAAAACCGTTTGCATATACTC-3′.






Nucleotides in lower caps are included as spacers just before the restriction sites between the endogenous TSHR and G protein. The sense and anti-sense primers included the restriction sites for XbaI and EcorV, respectively.

PCR was then utilized to secure the respective receptor sequences for fusion within the Gsα universal vector disclosed above, using the following protocol for each: 100 ng cDNA for TSHR(A623K) was added to separate tubes containing 2 μL of each primer (sense and anti-sense), 3 μL of 10 mM dNTPs, 10 μL of 10× TaqPlusTM Precision buffer, 1 μL of TaqPlusTM Precision polymerase (Stratagene: #600211), and 80 μL of water. Reaction temperatures and cycle times for TSHR were as follows: the initial denaturing step was done at 94° C. for five minutes, and a cycle of 94° C. for 30 seconds; 55° C. for 30 seconds; 72° C. for two minutes. A final extension time was done at 72° C. for ten minutes. PCR product for was run on a 1% agarose gel and then purified (data not shown). The purified product was digested with XbaI and EcorV (New England Biolabs) and the desired inserts isolated, purified and ligated into the Gs universal vector at the respective restriction site. The positive clones were isolated following transformation and determined by restriction enzyme digest; expression using Hek-293 cells was accomplished following the protocol set forth infra. Each positive clone for TSHR: Gs-Fusion Protein was sequenced and made available for the direct identification of candidate compounds. (See, SEQ.ID.NO.:588 for nucleic acid sequence and SEQ.ID.NO.:589 for amino acid sequence).
Location of non-endogenous version of TSHR(A623K) is located upstream from the rat G protein Gsα (i.e., from nucleotide 1 through 2,292; see, SEQ.ID.NO.:586 and amino acid residue 1 through 764; see, SEQ.ID.NO.:587). TSHR(A623K) can be linked directly to the G protein, or there can be spacer residues between the two. With respect to TSHR, 24 amino acid residues (an equivalent of 72 nucleotides) were placed in between the non-endogenous GPCR and the start codon for the G protein Gsα. Therefore, the Gs protein is located at nucleotide 2,365 through 3,549 (see, SEQ.ID.NO.:586) and at amino acid residue 789 through 1,183 (see, SEQ.ID.NO.:587). Those skilled in the art are credited with the ability to select techniques for constructing a GPCR Fusion Protein where the G protein is fused to the 3′ end of the GPCR of interest.
GPCR Fusion Protein was analyzed (to stabilize the GPCR while screening for candidate compounds, as shown in Example 6) and verified to be constitutively active utilizing the protocol found in Example 4(2). In FIG. 5, TSHR(A623K)-Gαs:Fusion Protein evidenced about an 87% increase in cAMP when compared to the control vector (pCMV).
2. GPR24-Giα Fusion Protein
GPR24-Giα Fusion Protein construct was then made as follows: primers were designed for both endogenous, constitutively activated and non-endogenous, constitutively activated GPR24 were as follows:









(SEQ. ID. NO.:584; sense)



5′-GTGAAGCTTGCCCGGGCAGGATGGACCTGG-3′



 



(SEQ. ID. NO.:585; anitsense)



5′-ATCTAGAGGTGCCTTTGCTTTCTG-3′.






The sense and anti-sense primers included the restriction sites for KB4 and XbaI, respectively.

PCR was then utilized to secure the respective receptor sequences for fusion within the Giα universal vector disclosed above, using the following protocol for each: 100 ng cDNA for GPR24 was added to separate tubes containing 2 μL of each primer (sense and anti-sense), 3 μL of 10 mM dNTPs, 10 μL of 10× TaqPlusTM Precision buffer, 1 μL of TaqPlusTM Precision polymerase (Stratagene: #600211), and 80 μL of water. Reaction temperatures and cycle times for GPR24 were as follows: the initial denaturing step was done it 94° C. for five minutes, and a cycle of 94° C. for 30 seconds; 55° C. for 30 seconds; 72° C. for two minutes. A final extension time was done at 72° C. for ten minutes. PCR product for was run on a 1% agarose gel and then purified (data not shown). The purified product was digested with KB4 and XbaI (New England Biolabs) and the desired inserts will be isolated, purified and ligated into the Gi universal vector at the respective restriction site. The positive clones was isolated following transformation and determined by restriction enzyme digest; expression using Hek-293 cells was accomplished following the protocol set forth infra. Each positive clone for GPR24: Gi-Fusion Protein was sequenced and made available for the direct identification of candidate compounds. (See, SEQ.ID.NO.:590 for nucleic acid sequence and SEQ.ID.NO.:591 for amino acid sequence).
Endogenous version of GPR24 was fused upstream from the G protein Gi and is located at nucleotide 1 through 1,059 (see, SEE.ID.NO.:588) and amino acid residue 1 through 353 (see, SEQ.ID.NO.:589). With respect to GPR24, 2 amino acid residues (an equivalent of 6 nucleotides) were placed in between the endogenous (or non-endogenous) GPCR and the start codon for the G protein Giα. Therefore, the Gi protein is located at nucleotide 1,066 through 2,133 (see, SEQ.ID.NO.:588) and at amino acid residue 356 through 711 (see, SEQ.ID.NO.:589). Those skilled in the art are credited with the ability to select techniques for constructing a GPCR Fusion Protein where the G protein is fused to the 3′ end of the GPCR of interest.
Although it is indicated above that Gi coupled receptors, such as GPR24, can be used in conjunction with a co-transfection approach, this is in the context of cAMP based assays and is predicated upon the effect of Gi on cAMP levels. However, for other types of assays, such as a GTP based assay, the co-transfection approach is not essential. Thus for assays such as a GTP based assay, the GPCR Fusion Protein approach is preferred such that, with respect to a GTP based assay for GPR24, the GPR24:Gi Fusion Protein would be preferred.
Example 6
Protocol: Direct Identification of Inverse Agonists and Agonists using [35S]GTPγS
Although Endogenous GPCRs may be utilized for the direct identification of candidate compounds as, e.g., inverse agonists, for reasons that are not altogether understood, intra-assay variation can become exacerbated. Preferably, then, a GPCR Fusion Protein, as disclosed above, can also be utilized with a non-endogenous, constitutively activated GPCR. We can determine that when such a protein is used, intra-assay variation appears to be substantially stabilized, whereby an effective signal-to-noise ratio is obtained. This has the beneficial result of allowing for a more robust identification of candidate compounds. Thus, it is preferred that for direct identification, a GPCR Fusion Protein be used and that when utilized, the following assay protocols be utilized.
1. Membrane Preparation
Membranes comprising the non-endogenous, constitutively active GPCR Fusion Protein of interest and for use in the direct identification of candidate compounds as inverse agonists, agonists or partial agonists are preferably prepared as follows:
a. Materials
“Membrane Scrape Buffer” is comprised of 20 mM HEPES and 10 mM EDTA, pH 7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1 mM EDTA, pH 7.4; “Binding Buffer” is comprised of 20 mM HEPES, 100 mM NaCl, and 10 mM MgCl2, pH 7.4.
b. Procedure
All materials will be kept on ice throughout the procedure. First, the media is aspirated from a confluent monolayer of cells, followed by rinse with 10 ml cold PBS, followed by a aspiration. Thereafter, 5 ml of Membrane Scrape Buffer will be added to scrape cells; this is followed by transfer of cellular extract into 50 ml centrifuge tubes (centrifuged at 20,000 rpm for 17 minutes at 4° C.). Thereafter, the supernatant is aspirated and the pellet is resuspended in 30 ml Membrane Wash Buffer followed by centrifugation at 20,000 rpm for 17 minutes at 4° C. The supernatant will then be aspirated and the pellet resuspended in Binding Buffer. This is then homogenized using a Brinkman PolytronTM homogenizer (15-20 second bursts until the all material is in suspension). This is referred to herein as “Membrane Protein”.
2. Bradford Protein Assay
Following the homogenization, protein concentration of the membranes will be determined using the Bradford Protein Assay (protein can be diluted to about 1.5 mg/ml, aliquoted and frozen (−80° C.) for later use; when frozen, protocol for use is as follows: on the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a Polytron at about 12×1,000 rpm for about 5-10 seconds; it is noted that for multiple preparations, the homogenizer should be thoroughly cleaned between homogenization of different preparations).
a. Materials
Binding Buffer (as per above); Bradford Dye Reagent; Bradford Protein Standard are utilized, following manufacturer instructions (Biorad, cat. no. 500-0006).
b. Procedure
Duplicate tubes will be prepared, one including the membrane, and one as a control “blank”. Each contained 800 μL Binding Buffer. Thereafter, 10 μL of Bradford Protein Standard (1 mg/ml) is added to each tube, and 10 μL of membrane Protein is then added to just one tube (not the blank). Thereafter, 200 μL of Bradford Dye Reagent is added to each tube, followed by vortex of each. After five (5) minutes, the tubes will be re-vortexed and the material therein is transferred to cuvettes. The cuvettes are then read using a CECIL 3041 spectrophotometer, at wavelength 595.
3. Direct Identification Assay
a. Materials
GDP Buffer consists of 37.5 ml Binding Buffer and 2 mg GDP (Sigma, cat. no. G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 μM GDP (final concentration of GDP in each well was 0.1 μM GDP); each well comprising a candidate compound, will have a final volume of 200 μL consisting of 100 μL GDP Buffer (final concentration, 0.1 μM GDP), 50 μL Membrane Protein in Binding Buffer, and 50 μL [35S]GTPγS (0.6 nM) in Binding Buffer (2.5 μL [35S]GTPγS per 10 ml Binding Buffer).
b. Procedure
Candidate compounds are preferably screened using a 96-well plate format (these can be frozen at −80° C.). Membrane Protein (or membranes with expression vector excluding the GPCR Fusion Protein, as control) will be homogenized briefly until in suspension. Protein concentration is then determined using the Bradford Protein Assay set forth above. Membrane Protein (and control) is then diluted to 0.25 mg/ml in Binding Buffer (final assay concentration, 12.5 μg/well). Thereafter, 100 μL GDP Buffer will be added to each well of a Wallac ScintistripTM (Wallac). A 5 μL pin-tool is then used to transfer 5 μL of a candidate compound into such well (i.e., 5 μL in total assay volume of 200 μL is a 1:40 ratio such that the final screening concentration of the candidate compound is 10 μM). Again, to avoid contamination, after each transfer step the pin tool should be rinsed in three reservoirs comprising water (1×), ethanol (1×) and water (2×)—excess liquid should be shaken from the tool after each rinse and dried with paper and kimwipes. Thereafter, 50 μL of Membrane Protein is added to each well (a control well comprising membranes without the GPCR Fusion Protein is also utilized), and pre-incubated for 5-10 minutes at room temperature. Thereafter, 50 μL of [35S]GTPγS (0.6 nM) in Binding Buffer will be added to each well, followed by incubation on a shaker for 60 minutes at room temperature (again, in this example, plates were covered with foil). The assay is then stopped by spinning of the plates at 4000 RPM for 15 minutes at 22° C. The plates will then be aspirated with an 8 channel manifold and sealed with plate covers. The plates are then read on a Wallacc 1450 using setting “Prot. #37” (per manufacturer instructions).
Example 7
Protocol: Confirmation Assay
Using an independent assay approach to provide confirmation of a directly identified candidate compound as set forth above, it is preferred that a confirmation assay then be utilized. In this case, the preferred confirmation assay is a cyclase-based assay.
A modified FLASH PLATETM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) is preferably utilized for confirmation of candidate compounds directly identified as inverse agonists and agonists to non-endogenous, constitutively activated GPCR in accordance with the following protocol.
Transfected cells will be harvested approximately three days after transfection. Membranes are prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl2. Homogenization is performed on ice using a Brinkman POLYTRONTM for approximately 10 seconds. The resulting homogenate will be centrifuged at 49,000×g for 15 minutes at 4° C. The resulting pellet is then resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000×g for 15 minutes at 4° C. The resulting pellet can be stored at −80° C. until utilized. On the day of direct identification screening, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl2, to yield a final protein concentration of 0.60 mg/ml (the resuspended membranes are placed on ice until use).
cAMP standards and Detection Buffer (comprising 2 μCi of tracer [125I cAMP (100 μl)] to 11 ml Detection Buffer) will be prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer will be prepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mM MgCl2, 20 mM phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase (Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer can be stored on ice until utilized.
Candidate compounds identified as per above (if frozen, thawed at room temperature) will then be added, preferably, to 96-well plate wells (3 μl/well; 12 μM final assay concentration), together with 40 μl Membrane Protein (30 μg/well) and 50 μl of Assay Buffer. This mixture is then incubated for 30 minutes at room temperature, with gentle shaking.
Following the incubation, 100 μl of Detection Buffer is added to each well, followed by incubation for 2-24 hours. Plates are then counted in a Wallac MICROBETATM plate reader using “Prot. #31” (as per manufacturer instructions).
Example 8
Ligand-Based Confirmation Assay
Membranes will be prepared from transfected Hek-293 cells (see Example 3) by homogenization in 20 mM HEPES and 10 mM EDTA, pH 7.4 and centrifuged at 49,000×g for 15 min. The pellet will be resuspended in 20 mM HEPES and 0.1 mM EDTA, pH 7.4, homogenized for 10 sec using Polytron homogenizer (Brinkman) at 5000 rpm and centrifuged at 49,000×g for 15 min. The final pellet will be resuspended in 20 mM HEPES and 10 mM MgCl2, pH 7.4, homogenized for 10 sec using Polytron homogenizer (Brinkman) at 5000 rpm.
Ligand-based confirmation assays will be performed in triplicate 200 μl volumes in 96 well plates. Assay buffer (20 mM HEPES and 10 mM MgCl2, pH 7.4) will be used to dilute membranes, tritiated inverse agonists and/or agonists and the receptor's endogenous ligand (used to define non-specific binding). Final assay concentrations will consist of 1 nM of tritiated inverse agonist and/or agonist, 50 μg membrane protein (comprising the receptor) and 100 μm of endogenous ligand. Agonist assay will be incubated for 1 hr at 37° C., while inverse agonist assays are incubated for 1 hr at room temperature. Assays will terminate by rapid filtration onto Wallac Filtermat Type B with ice cold binding buffer using Skatron cell harvester. The radioactivity will be determined in a Wallac 1205 BetaPlate counter.
Again, this approach is used merely to understand the impact of the directly identified candidate compound on ligand binding. As those in the art will appreciate, it is possible that the directly identified candidate compounds may be allosteric modulators, (i.e., compounds that affect the functional activity of the receptor but which do not inhibit the endogenous ligand from binding to the receptor. Allosteric modulators include inverse agonists, partial agonists and agonists.
References cited throughout this patent document, including co-pending and related patent applications, unless otherwise indicated, are fully incorporated herein by reference. Modifications and extension of the disclosed inventions that are within the purview of the skilled artisan are encompassed within the above disclosure and the claims that follow.
Although a variety of expression vectors are available to those in the art, for purposes of utilization for both the endogenous and non-endogenous known GPCRs, it is most preferred that the vector utilized be pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd., Manassas, Va. 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be. The ATCC has assigned the following deposit number to pCMV: ATCC #203351.

## Claims

1. A method for directly identifying a non-endogenous compound as a compound having inverse agonist, agonist, or partial agonist activity to a non-endogenous, constitutively activated version of a known G protein-coupled receptor, wherein said non-endogenous, constitutively activated version comprises the amino acid sequence of SEQ.ID.No.:471, said method comprising:
(a) contacting a non-endogenous candidate compound with said non-endogenous, constitutively activated version of the known GPCR comprising the amino acid sequence of SEQ.ID.No.:471; and
(b) measuring the compound efficacy at said contacted receptor, and identifying whether said non-endogenous compound has inverse agonist activity, agonist activity, or partial agonist activity at said contacted receptor.

2. The method of claim 1 further comprising the following step:
assessing, by using an endogenous ligand based assay, the effect of the non-endogenous compound identified as having inverse agonist, agonist, or partial agonist activity on the binding of an endogenous ligand to said known GPCR.

3. The method of claim 2 wherein said non-endogenous compound is an allosteric modulator of said known GPCR.