Somatostatin receptor protein

The present invention relates to a method for isolating and cloning receptor DNA sequences. The invention also provides novel DNA sequences encoding a novel somatostatin receptor subtype.

The superfamily of G protein-linked receptors controls many physiological 
functions. These receptors mediate transmembrane signaling from external 
stimuli (vision, taste and smell), endocrine function (pituitary and 
adrenal), exocrine function (pancreas), heart rate, lipolysis, and 
carbohydrate metabolism. The molecular cloning of only a fraction of the 
genes for this family has revealed many structural and genetic 
similarities including seven membrane spanning domains, conserved amino 
acids critical for function, and sites for glycosylation and 
phosphorylation. In many cases, the genes for these receptors lack 
introns, a rarity among eukaryotic genes. The G protein-linked receptor 
superfamily can be subclassified into five distinct groups: (i) amine 
receptors (serotonin, adrenergic, etc.); (ii) small peptide hormone 
(somatostatin, TRH, etc.); (iii) large peptide hormone (LH-CG, FSH, etc.); 
(iv) secretin family; and (v) odorant receptors (2). The isolation of as 
yet uncloned receptors that are associated with important physiological 
functions or disease states, or which are critical for enhancement of 
animal performance e.g., receptors for growth hormone release factor, 
corticotropin release factor, cholecystokinin, vasoactive intestinal 
peptide (high affinity subtype)! would be highly beneficial. 
Current strategies for cloning members of this receptor superfamily are 
diverse and fraught with difficulties. These techniques require the 
isolation of a full-length cDNA or gene to determine whether a particular 
protein is actually the receptor of interest. For example, expression 
cloning using a heterologous system (such as Xenopus oocytes) requires the 
isolation of the full-length cDNA (15). The use of PCR with degenerate 
oligonucleotides has also been used to clone G protein-linked receptors. 
However, this approach has been largely applied only to amine receptors, 
and not to peptide hormone receptors (9). A major drawback to that 
methodology is the generation of "orphan" receptors where the 
identification of the receptor, via identification of the hormone that 
activates it, may remain unknown for years. Finally, protein purification 
is difficult because these receptors are, in general, very low in 
abundance. Obtaining peptide sequence data is, therefore, an arduous and 
time-consuming task. In contrast, the present application describes a 
system that does not require isolation of a full-length cDNA until a PCR 
fragment has already been identified as originating from that gene. 
Additionally, utilization of the highly processive PCR technique, in 
combination with other steps, circumvents any difficulties due to low 
abundance. 
SUMMARY OF THE INVENTION 
The present invention relates to a method for isolation and identification 
of novel receptor DNA. The method comprises steps of (a) contacting a 
template nucleic acid, in a polymerase chain reaction, with at least one 
target receptor-specific oligonucleotide primer, and isolating a nucleic 
acid fragment resulting from the reaction; (b) providing an antisense 
oligonucleotide based on a nucleotide sequence from the isolated fragment 
to a cell capable of expressing a particular receptor, and (c) observing 
the presence or absence of the particular receptor's expression or 
function, in that cell. The absence of receptor expression indicates that 
the antisense oligo has blocked that receptor's expression, thereby 
confirming the identity of the isolated nucleic acid fragment upon which 
the antisense oligo was based as encoding all or a portion of the receptor 
whose expression has just been measured. 
The present method is useful for identifying receptors from virtually any 
class, but is particularly useful in identification and isolation of G 
protein-linked receptor. For use in identification of this receptor type, 
the invention also provides a series of several novel oligonucleotides 
useful as primers in the first step polymerase chain reaction. The method 
has also resulted in the identification of a heretofore unknown 
somatostatin receptor subtype, herein designated as SSTR3; the invention 
therefore also provides a nucleic acid sequence encoding that receptor, as 
well as vectors and host cells capable of expressing the sequence. 
The present invention provides significant advantages over known methods of 
receptor sequence isolation and cloning. It provides an alternative to the 
process of isolating receptor protein in order to obtain sequence 
information, a task which is necessarily arduous because of the frequently 
small amounts of expressed receptor in most cells; the use of PCR in the 
present method overcomes this problem. The ease of identification of the 
present method also avoids the classic problem of "orphan receptors", 
i.e., isolated nucleic acid fragments presumed to encode a receptor 
protein, but the identity of which remains unknown for lack of a reliable 
assay and/or a complete sequence. The claimed method provides a means for 
immediately identifying the ligand specificity of the isolated fragment by 
quick assay of its activity in wild-type cells and further avoids the 
necessity for having a full-length cDNA clone for heterologous expression 
in a host cell.

DETAILED DESCRIPTION OF THE INVENTION 
The present method can be employed to isolate and clone nucleotide 
sequences encoding proteins from any family of proteins for which (1) a 
consensus sequence or sequences can be identified; (2) a cell line 
expressing the protein is available; and (3) a functional assay exists. 
However, the method is particularly useful in isolation of the nucleotide 
sequences encoding various types of receptors. Although the specific 
examples provided in this application relate to isolation and 
identification of a nucleic acid fragment encoding a G protein-linked 
receptor for a small peptide hormone, those skilled in the art will 
readily recognize how the techniques described herein can be applied to 
other receptors as well. 
The first step of the method employs the polymerase chain reaction 
(hereinafter "PCR") to amplify the amount of receptor nucleic acid 
available for study. The nucleic acid used in the PCR is not restricted to 
any particular source, but the source cells will preferably be obtained 
from tissue which is known to express a particular receptor or receptors 
of interest. Tissue sources for various receptors are well known in the 
art, as seen, for example in Trends in Pharmacological Sciences, Receptor 
Supplement, Jan. 1992 (24). Once an appropriate target cell or tissue type 
is identified, genomic DNA, cDNA, either from a library or synthesized 
from RNA, or RNA itself is isolated therefrom by standard methods (20), in 
preparation for use as a template for PCR. 
The technique of PCR is described in U.S. Pat. Nos. 4,683,202; 4,683,195; 
and 4,965,188; the contents of these patents are incorporated herein by 
reference. In brief, PCR is useful in amplifying, cloning and/or detecting 
a target sequence in a sample of DNA. Typically, the native duplex DNA 
strand is denatured, so that the individual strands separate. The separate 
strands each constitute a template which is contacted with primers, which 
are usually oligonucleotides designed to be able to anneal with a portion 
of the target sequence contained on the template. The primers of each 
strand are then extended by the use of DNA polymerase, thereby providing 
two new copies of the target sequence. The entire process is repeated 
numerous times, thus potentially resulting in millions of copies of the 
targeted sequence, making detection of a gene expressed at very low levels 
considerably easier. 
As applied to the present method, the template nucleic acid is derived from 
a cell known or expected to carry nucleic acid encoding one or more 
receptors of interest. The nucleic acid is then contacted with primers 
(oligos) specifically designed to anneal with sequences characteristic of 
the receptor or receptors of interest. For example, in the examples 
provided herein, comparison of the known nucleotide sequences (see 
generally 19), particularly of somatostatin (25), substance K (23), 
substance P (23, 6, 26, 4, 21), neuromedin K (22), thyrotropin (8, 9, 17, 
19), LH/hCG (12, 14, 16), and other receptors designated mas (27), mrg and 
rta indicates from regions exhibiting a significant degree of 
conservation. From this observation, five oligonucleotide primers are 
designed; with 4- to 64-fold degeneracy plus 5-23% inosines. (See FIG. 
2A.) These oligos are capable of recognizing virtually any receptor for a 
peptide ligand in the PCR procedure, and indeed, as shown below, are used 
to isolate the sequence encoding a novel somatostatin receptor. Other 
heretofore unisolated receptor genes which may be cloned using these 
oligos include, but are not limited to, the receptors, CRF (corticotropin 
release) GNRH (gonadotropin release hormone), follitropin release hormone, 
growth hormone releasing hormone, octopamine, galanin, adenosine subtypes, 
angiotensin (II) subtype monocyte chemoattractant protein-1, and 
vasopressin subtypes. 
If a different type of receptor is sought, alternate oligos can readily be 
constructed. For example, to identify genes encoding the secretin type of 
receptor (which are structurally similar to the peptide hormone receptors, 
but have quite distinct amino acid sequences), a comparison of the 
secretin, calcitonin and parathyroid receptor gene sequences provide the 
information for design of four novel oligos which can be used in 
recognition of receptors of this subclass (FIG. 2B). This group of 
receptors potentially includes secretin, calcitonin, parathyroid hormone, 
growth hormone releasing factor (GRF) and glucagon. 
For the identification of amine receptors, a number of oligos have already 
been described (8). Similarly, appropriate sequences for odorant receptors 
have also been described (2). In each case, these can be used in the same 
manner as the oligos described above. 
The foregoing lists relate to G protein-linked receptors, but oligos for 
use in this method can also be made for other types of receptors, e.g., 
non G protein-linked peptide hormone receptors and steroid hormone 
receptors. The appropriate oligonucleotides can be designed by comparison 
of several known related genes of that receptor type to determine regions 
of homology among them, then creating degenerate oligos based on one or 
more of these consensus sequences. 
In the PCR procedure, the number of primers used is not critical and, to 
some extent, will be governed by the number of useful oligos available for 
this purpose. Generally, optimal primer number is determined empirically, 
dependent on the results obtained with a first pair of primers used. 
Typically, a balance is struck between the number of oligo pairs, the 
number of homologous sequences present in each receptor subclass to which 
oligos may be directed, and the facilities available to analyze the 
products. 
The conditions for PCR reactions are well known in the art. The resulting 
clones are then isolated and the nucleotide sequence determined for all or 
part of the clone. At this point, the clone still represents an "orphan", 
the identity of which remains to be determined. However, it can be 
determined at this time whether the peptide encoded by the clone has the 
general hallmarks of a receptor molecule, particularly of the receptor 
type originally selected for. For example, membrane bound receptors are 
arranged in a pattern of intracellular (IC), external and transmembrane 
(TM) domains, though the types of these domains are specific to each group 
of receptors. The presence of the characteristic domains can be determined 
from the predicted amino acid sequence by analysis by the Kyte-Doolittle 
hydropathy index (7; See also FIG. 4). Once the protein has been confirmed 
as likely being a receptor, its subclass identity can be somewhat further 
refined before proceeding to the next stage of the process. A relatively 
short (i.e., 8-23 amino acids) third intracellular loop (IC3), for 
example, is characteristic of G protein-linked peptide ligand receptors; 
in contrast, the IC3 of amine receptors generally has about 47-155 
residues, and contain a conserved Asp residue in their third transmembrane 
domain. Although this determination is not essential to the success of the 
method, it will help to screen out clones which are not likely to have 
been derived from receptor DNA, and to select for those that are likely to 
be receptor-derived. 
In order to more specifically determine the identity of the receptor, the 
sequence obtained from selected clones is then used to design antisense 
oligonucleotides. The specific length of the oligo is not critical, but it 
is preferable to use the largest oligo that can be taken up by cells to 
provide the greatest sequence specificity. Typically oligos comprise 15 to 
21 nucleotides. Generally, at least two complementary oligonucleotides are 
made, one in the sense orientation and one in the antisense orientation. 
Preferably the antisense oligos are designed to correspond to regions of 
mRNA that display the least amount of predicted secondary structure. Each 
oligo preferably contains 50-70% cytosines plus guanines to maximize 
hybridization to mRNA. The antisense oligo is used to evaluate whether any 
receptor function can be blocked in a wild-type cell. If the orphan clone 
from which the antisense sequence is derived does represent all or part of 
a receptor gene, then the antisense oligo (but not the sense oligo), when 
provided to a cell normally capable of expressing that same receptor, 
should block expression of that receptor. 
Since the desired subclass of receptor is determined at the start of the 
procedure, the type of cell used to evaluate the effect of the antisense 
oligo will be selected from among cells known to normally express 
receptors of the type and subclass initially sought. Whole cells or cell 
membranes from the selected cell type are treated with either an antisense 
oligo, its complementary sense oligo, or vehicle (as a control). The 
treated preparations are then observed to determine the presence or 
absence of receptor function in the presence of a ligand which normally 
binds to the receptor. In a typical situation, a variety of ligands are 
tested; the ligands selected for testing are those which are known to bind 
to receptors within the subclass sought. 
The activity of a receptor in the presence of its ligand can be determined 
in a number of ways. The ligand can be detectably labelled, e.g., with a 
radioisotope, chemiluminescent molecule, or any other detectable label 
commonly used in the art. The labelled ligand is then contacted with the 
treated cells, or preferably cell membranes from treated cells, and the 
presence or absence of binding of the ligand to the cell or cell membranes 
determined. The observation of ligand binding in an antisense-treated cell 
indicates that the antisense oligo had no effect on expression of that 
ligand's receptor, thereby eliminating that receptor from consideration as 
being the one encoded by the orphan clone. On the other hand, the absence 
or reduction of bound ligand to cell membranes from antisense treated 
cells, combined with the observation of ligand binding in the 
sense-treated cell membranes, indicates that the original orphan clone 
represents all or a portion of the gene encoding the receptor for that 
particular ligand. 
Alternately, assuming that the receptors sought are of the type whose 
action is mediated by a second messenger, then receptor function can be 
evaluated by measuring accumulation of the second messenger. For example, 
many peptide hormones mediate their ultimate effects by way of the action 
of cyclic AMP (cAMP). Thus, the binding of a particular ligand to a 
receptor can be evaluated by measuring the accumulation of cAMP within 
antisense-treated intact cells. If there is no stimulation or inhibition 
(depending on the receptor) of cAMP accumulation in an antisense-treated 
cell in the presence of a particular ligand, then it can be assumed that 
the receptor for that ligand was not expressed, and that the orphan 
sequence shares identity with the receptor for that ligand. Cyclic cAMP 
accumulation assays are well known in the art (3, 5). However, the 
practice of the invention is not limited to identification of receptors 
for peptide hormones where activity is mediated by cAMP. Other second 
messenger systems are also known, e.g., ion channels are detected by patch 
clamp, and inositol phosphates and arachidonic acid can be assayed using 
commercially available kits (New England Nuclear). In the case in which 
second messenger accumulation is used as the central indication of 
receptor blockage, labelled ligand binding can be used as a further 
confirmation of receptor identity. 
If it should be the case that the original clone represents only a fragment 
of the receptor, further screening is necessary to isolate the entire 
gene. This can be easily achieved at this stage, however, because the 
fragment has already been conclusively demonstrated to be 
receptor-derived, and therefore can be reliably utilized as a probe to 
screen genomic or cDNA libraries for the entire receptor sequence. 
Alternately, a method for rapid amplification of cDNA ends (RACE; 11) 
permits cloning of a complete gene sequence without library screening. 
Sequencing and subsequent cloning of the entire sequence can be achieved 
using standard techniques available in the art (20). Utilizing the 
foregoing method, a novel somatostatin receptor is identified. 
It will be understood by those skilled in the art that the present method 
is applicable to a wide variety of receptors. Among the receptors of 
particular interest are the receptors for GRP, VIP, galanin, glucagon, 
.beta.-endorphin, CCKB, GHRH, GNRH, follitropin releasing hormone, CRF, 
octopamine, adenosine subtypes, angiotensin II subtypes, monocyte 
chemoattractant protein-1, and vasopressin isoforms. There are also 
convenient, publicly available host cell lines for use in the antisense 
assay: for example, Rin cells provide a source of expressed glucagon and 
galanin receptors; RC-4B cells are a useful cell line for GRF, GHRH, GNRH, 
and follitropin releasing factor receptors. NG108 cells are a source of 
.beta.-endorphin receptor. AtT20 cells can be used in connection with CRF 
receptor. Neuroblastoma cells are a convenient source for CCKB. 
In addition to those receptors and cell lines noted above, variations of 
the claimed method will also be apparent to those skilled in the art. 
The invention is further illustrated in the following non-limiting 
examples. 
EXAMPLES 
1. General Methods 
Unless otherwise specified, the following methods are utilized in the 
procedures described below: 
Polymerase Chain Reaction. Oligonucleotides are synthesized on an Applied 
Biosystems Model DNA synthesizer. PCR reactions are done with the 
Perkin-Elmer Cetus GeneAmp.RTM. PCR kit if using DNA as the template or 
with the same manufacturer's GeneAmp.RTM. rTth RNA PCR kit if using RNA as 
the template. Reactions are performed, but not limited to, the 
manufacturer's recommendations with a 39.degree. C. annealing temperature 
for degenerate oligonucleotides or 60.degree. C. for specific primers. 
Each reaction contains 1 .mu.g DNA or total cellular RNA plus 1 .mu.g of 
each primer (ca. 1 .mu.M final concentration). 
Sequence Analysis. PCR products are separated in a 1.2% agarose slab gel 
containing 1 .mu.g/ml ethidium bromide. DNA fragments of the expected 
length are cut out and purified with GeneClean.RTM. (BIO101). The termini 
are polished with T4 DNA polymerase in the presence of 0.25 mM dNTPs and 
ligated into pGEM3Z (Promega Corp.) cleaved with SmaI. Selected clones are 
sequenced using the Applied Biosystems dye primer DNA sequencing kit and 
the Applied Biosystems Model 373A automated sequencer. Protocols are as 
recommended by Applied Biosystems. Nucleotide sequences are analyzed with 
the MacVector.RTM. software package. 
2. Isolation of Receptor Fragments 
To target peptide receptors, consensus sequences are identified from nine 
rat genes. These sequences are then utilized to design probes for the 
isolation of new genes of the peptide receptor class. The receptors used 
are those for somatostatin, substance K, substance P, neuromedin K, 
thyrotropin, LH/CG and others designated mas, mrg, and rta. Four regions 
of nucleotide sequence are found to exhibit a degree of conservation 
significant enough to design degenerate oligonucleotide primers for PCR. 
Five oligos are designed with 4- to 64-fold direct degeneracy plus 5% to 
23% inosines (FIG. 2A). Additional probes are developed for the class of G 
protein-linked receptors characterized by the rat secretin receptor. This 
class currently has only four known members (recognizing secretin, 
calcitonin, parathyroid hormone and vasoactive intestinal peptide). The 
sequences for these genes are aligned and four oligonucleotides are 
subsequently designed to potentially recognize this subtype (FIG. 2B). 
Neither set of oligos would be predicted to amplify amine receptor 
sequences. 
The five oligos described in FIG. 2A are designed to anneal sequences in G 
protein-linked peptide receptor genes. Oligos are synthesized on an 
Applied Biosystems Model DNA synthesizer. The oligos are used as six 
different primer pairs for PCR. PCR reactions are conducted as described 
above. Using rat genomic DNA as the template, all six reactions produce 
DNA fragments of the expected size (ranging from ca. 420 bp for a tm3 to 
tm6 fragment to ca. 700 bp for a tm2 to tm7 fragment), DNA fragments are 
cloned and two samples, designated PCR9 and PCR11, are found to contain 
sequences characteristic of G protein-linked receptors but no exact 
matches are found in GenBank (Table 1). Nucleotide sequences of these two 
fragments are shown in FIG. 3. Each fragment contains a single open 
reading frame. The comparison with other receptor sequences shows a 
considerable similarity between a rat somatostatin receptor and PCR 11, 
suggesting that PCR11 is a novel somatostatin receptor subtype. Both 
fragments exhibit not only primary sequence similarity to other receptors 
but also the hydropathy pattern characteristic of G protein-linked 
receptors (FIG. 4). Interestingly, the predicted third intracellular loop 
(IC3) of each new sequence is comparatively short, a hallmark of peptide 
receptors which have IC3s of 8 to 23 amino acids long (PCR #9 has an IC3 
of 13 residues and PCR #11 has one of 23 amino acids). In contrast, the 
IC3s of amine receptors range from 47 to 155 residues in length. These 
observations suggest that the novel G protein-linked receptors represented 
by the PCR products are of the peptide ligand subclass. 
TABLE 1 
______________________________________ 
Relatedness of Novel PCR Fragments 
to G Protein-Linked Receptors.sup.a 
Gene 
Receptor Class 
Designation.sup.b 
PCR #9.sup.c 
PCR #11.sup.c 
______________________________________ 
Peptide Ligand 
srif 37 57 
neuK 24 30 
subK 22 25 
subP 20 26 
mas 23 16 
mrg 15 18 
thy 19 18 
lhcg 16 13 
Amine Ligand 
.alpha.1b 21 27 
.beta.2 24 20 
d2 10 22 
______________________________________ 
Notes: 
.sup.a Each PCR fragment (designated #9 or #11) is translated and compare 
to G proteinlinked receptors available in GenBank. 
.sup.b All sequences are from rat. Designations are: srif, somatostatin 
receptor; neuK, neuromedin K receptor; subK or P, substance K or P 
receptor; mas, mas oncogene; mrg, mas related gene; thy, thyrotropin 
receptor; lhcg, luteinizing hormone and chorionic gonadotropin receptor; 
.alpha.1b, .alpha..sub.1badrenergic receptor; .beta.2, 
.beta..sub.2adrenergic receptor; d2, D.sub.2 dopamine receptor. 
.sup.c Numbers represent the percent identical or conserved amino acids 
over the length of the translated PCR fragment. Any percentage greater 
than 15 is considered highly significant. 
3. Blockade of Somatostatin Receptor and Expression Expression Function 
with Antisense Oligos 
A. Ligand Binding Assay 
GH4C.sub.1 (a pituitary cell line) cells are grown to 50% confluence in 10% 
fetal bovine serum, Dulbecco's modified Eagles medium in a humidified 
chamber (37.degree. C., 95:5, air CO.sub.2). The GH.sub.4 C.sub.1 cells 
are then incubated with vehicle (H.sub.2 O) (control), with an antisense 
oligo (18 mer, GGGTCGCCTCCATTTCGG, 5 .mu.M (SEQ ID NO:5)), or the 
complementary sense strand (18 mer, GCCAAATGGAGGCGACCC, 5 .mu.M (SEQ ID 
NO:6)) for 4 hours at 37.degree. C. (95:5, air CO.sub.2) in Dulbecco's 
modified Eagles medium without serum. At the end of the 4-hour time 
period, heat-inactivated fetal bovine serum is added to 10% (final 
concentration). The cells are grown for an additional 20 hours. The cells 
are harvested, crude membranes are prepared as described by Eppler et al. 
(28). Radioligand binding is performed with .sup.125 I!tyr-somatostatin, 
S-14 (50 fmol/tube, 250 pMol final), using standard binding assay methods 
as follows: binding assays are done in a binding buffer containing 50 mM 
HEPES (pH 7.4), 0.5% BSA and 5 mM MgCl.sub.2. The standard filtration 
assay for .sup.125 I!SRIP analog binding to GH.sub.4 C.sub.1 membranes is 
carried out as follows: 1. Radioligand is diluted in binding 
buffer+PMSF/Baci to the desired cpm per vol. of 10 .mu.l and then 180 
.mu.l aliquots are added to the table. For non-specific binding samples, 
10 .mu.l of 20 .mu.M cold S14 is also added per well of membrane diluted 
to the desired concentration (10-30 ug membrane protein/well) in binding 
buffer+PMSF/Baci. Binding is stopped by rapid filtration of samples 
through Whatman GF/C filters presoaked with 0.3% polyethyleneimine. 
Binding is allowed to proceed at 30.degree. C. for 1-2 hours. Binding is 
stopped by rapid filtration of samples through Whatman GF/C filters 
presoaked with 0.3% polyethyleneimine. Each sample is washed three times 
with ice-cold binding buffer (5 ml). Finally the individual wells are 
placed in 12.times.75 mm tubes and counted in an LKB Gammamaster counter 
(78% efficiency). The results, displayed in FIG. 5, are the average of two 
experiments, each performed in triplicate. It can be seen that membrane 
from cells treated with vehicle and the sense oligo display equivalent 
levels of binding .sup.125 I!tyr-somatostatin (408 v 350 fmol/mg). In 
membranes challenged with the antisense oligo for 24 hours, a 60% decline 
in .sup.125 I!tyr-somatostatin binding is observed (166 mol/mg). Thus 
blockade of receptor expression with antisense, but not the sense oligos 
shows a specific means by which loss of function can be used to 
characterize and identify orphan receptors. 
B. cAMP Assay 
AtT20 cells are grown to 50% confluence in 10% fetal bovine serum, 
Dulbecco's modified Eagles medium in a humidified chamber (37.degree. C., 
90:10, air:CO.sub.2). The AtT20 cells are then incubated with vehicle 
(control), with an antisense oligo directed against the orphan G 
protein-linked receptor PCR11 (20 mer, CGAGCGCCTCCGCCTTGAGG, 5 .mu.M (SEQ 
ID NO:7)), or the sense strand (20 mer, ACGCAGAACGCCGTCTCCTA, 5 .mu.M (SEQ 
ID NO:8)) for 4 hours at 37.degree. C. (90:10, air:CO.sub.2) in Dulbecco's 
modified Eagles medium without serum. The mRNA encoding this orphan 
receptor is detected in AtT-20 mouse pituitary cells by PCR Northern 
analysis. At the end of the four-hour time period, heat-inactivated fetal 
bovine serum is added to 10% (final concentration). The cells are 
harvested and bovine serum is added to 10% (final concentration). The 
cells are harvested and resuspended in Kreb's Ringer phosphate buffer 
containing 2 mM CaCl.sub.2, 100 .mu.M isobutyl-methylxanthine (to inhibit 
cyclic AMP-dependent phosphodiesterase activity). Somatostatin-mediated 
inhibition of forskolin-stimulated cyclic AMP accumulation is performed in 
intact cells. Cyclic AMP is measured using a kit purchased from Amersham. 
Cells are washed two times with Kreb's Ringer Phosphate (KRP) buffer 
containing 1 m CaCl.sub.2. The cells (50-100,000/tube in triplicate) are 
incubated for 15 minutes with activators and/or inhibitors of adenylyl 
cyclase in KRP buffer (final vol=100 .mu.l) containing 100 .mu.M 
isobutyl-methylxanthine. The reaction is terminated by addition of 10 
.mu.l of 0.1 N HCl. The samples are boiled for 5 minutes and then 
neutralized with 0.1 N NaOH containing 100 mM Tris, pH 7.5. To each tube 
.sup.3 H! cyclic AMP is added (-15,000 cpm) and 10 .mu.l of adrenal 
binding protein. The samples are incubated on ice for 90 minutes with 
standards, activated charcoal (100 .mu.l) is added, and centrifuged 
(4.degree. C., 2000.times.g for 5 minutes). The samples are compared 
against standards of known concentrations and quantified by liquid 
scintillation spectrometry. Results are depicted in FIG. 6. The figure 
shows that the efficacy of somatostatin mediated inhibition of cAMP 
accumulation is attenuated. Also, the EDSO for the somatostatin medicated 
response is shifted to the right. In control and sense treated cells, the 
ED50s for somatostatin receptor are calculated to be 1 and 1.2 nM, 
respectively. In the cells treated with antisense oligos, the ED50 
response to somatostatin is 9 nM, a 9-fold shift in potency. These data 
indicate that both the efficacy and potency of somatostatin are altered by 
treatment of cells with antisense oligos. Thus, the PCR11 fragment appears 
to identify a novel somatostatin receptor subtype. The fragment is 
compared with known receptor subtypes SSTR1 and SSTR2 (25). Over the 
region currently available (representing greater than 50% of the coding 
sequence), the SSTR3 receptor is 57% identical to SSTR2, and is 74% 
homologous if conservative amino acid substitutions are considered (FIG. 
8); SSTR3 is only slightly less homologous to SSTR1: 49% identical and 70% 
identical or conserved residues. 
In the course of further experiments, a mouse homologue is also isolated. 
The nucleotide sequence of this fragment is given in FIG. 7. 
DEPOSIT OF BIOLOGICAL MATERIALS 
The following materials have been deposited on Jun. 4, 1992, under the 
terms of the Budapest Treaty, at the American Type Culture Collection, and 
given the indicated accession numbers. 
______________________________________ 
Description Accession No. 
______________________________________ 
E. coli K-12, 0Z9 (PCR9) 
69005 
E. coli K-12, 0Z11 (PCR11) 
69006 
______________________________________ 
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24. Trends in Pharmacological Sciences. Receptor Supplement, Elsevier, Jan. 
1992. 
25. Yamada, Y. et al., PNAS USA, 89:251-255, 1992. 
26. Yokotu, Y. et al., J. Biol. Chem., 264:17649-17652, 1989. 
27. Young, D., PNAS USA, 85:5339-5342, 1988. 
28. Eppler, C. M. et al., J. Biol. Chem., (in press), 1992. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 19 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 545 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Rat 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- TTCGTGGTGA ACCTGGTCGG GGCTGACTTT CTCCTGATCA TTTGCTTGCC GT - #TCTTGACG 
60 
- GACAACTATG TCCAGAACTG GGACTGGAGG TTCGGGAGCA TCCCCTGCCG CG - #TGATGCTC 
120 
- TTCATGTTGG CCATGAACCG ACAGGGCAGC ATCATCTTCC TCACGGTGGT GG - #CTGTGGAC 
180 
- AGGTACTTCA GGGTGGTCCA CCCGCACCAC TTCCTGAACA AGATCTCCAA CC - #GGACGGCG 
240 
- GCCATCATCT CTTGCTTCCT GTGGGGCATC ACCATCGGCC TGACAGTCCA CC - #TCCTCTAC 
300 
- ACGGACATGA TGACCCGAAA CGGCGATGCA AACCTGTGCA GCAGTTTTAG CA - #TCTGCTAC 
360 
- ACTTTCAGGT GGCACGATGC AATGTTCCTC TTGGAATTCT TCCTGCCCCT GG - #GCATCATC 
420 
- CTGTTCTGCT CTGGCAGGAT CATTTGGAGC CTAAGGCAGA GACAGATGGA CA - #GGCACGTC 
480 
- AAGATCAAGA GGGCCATCAA CTTCATCATG GTGGTTGCCA TTGTGTTTGC CA - #TCTGCTGG 
540 
# 545 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 563 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Rat 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- TTCGTGCTGC ACCTGGTCGG GGCTGACGTA TTATTTATGT TGGGACTTCC TT - #TCCTGGCC 
60 
- ACGCAGAACG CCGTCTCCTA CTGGCCCTTC GGCTCCTTCT TGTGCCGCCT GG - #TCATGACA 
120 
- CTGGATGGCA TCAACCAGTT CACCAGTATC TTCTGCCTGA TGGTCATGAG TG - #TTGACCGC 
180 
- TACCTGGCCG TGGTCCACCC TCTCCGCTCA GCCCGGTGGC GTCGCCCACG GG - #TAGCCAAG 
240 
- ATGGCCAGCG CGGCCGTCTG GGTCTTTTCG CTGCTCATGT CTCTGCCGCT CT - #TGGTCTTC 
300 
- GCGGATGTCC AGGAGGGCTG GGGCACCTGC AACCTGAGCT GGCCAGAGCC TG - #TGGGGCTG 
360 
- TGGGGTGCAG CCTTCATCAC CTACACGTCT GTGTTGGGCT TCTTTGGGCC CC - #TGCTGGTC 
420 
- ATCTGCTTGT GCTACCTGCT CATTGTGGTC AAGGTGAAGG CTGCAGGCAT GC - #GCGTAGGC 
480 
- TCCTCAAGGC GGAGACGCTC GGAGCGCAAG GTGACTCGCA TGGTGGTGGT CG - #TGGTGCTG 
540 
# 563GGCT GCC 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 678 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Mouse 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- TTCGTGCTGA ACCTGGCCGG GGCTGACGTG TTGTTTATGT TGGGGCTTCC TT - #TCCTGGCA 
60 
- ACGCAGAATG CTGTCTCCTA CTGGCCCTTT GGCTCCTTCT TGTGCCGCCT GG - #TCATGACG 
120 
- CTGGACGGCA TCAACCAGTT CACCAGTATC TTCTGCCTGA TGGTCATGAG TG - #TCGACCGC 
180 
- TACCTGGCCG TGGTCCACCC TCTCCGCTCA GCCCGGTGGC GTCGCCCACG GG - #TAGCCAAG 
240 
- CTGGCTAGTG CTGCCGTCTG GGTCTTCTCG CTGCTCATGT CTCTGCCGCT CT - #TGGTCTTT 
300 
- GCGGATGTCC AGGAGGGCTG GGGCAACTGC AACCTGAGCT GGCCAGAGCC TG - #TGGGAATG 
360 
- TGGGGTGCAG CCTTCATCAC TTACACGTCT GTGCTGGGCT TCTTTGGGCC CC - #TGCTGGTC 
420 
- ATCTGCATGT GCTATTTGCT CATCGTAGTG AAGGTGAAGG CTGCAGGTAT GC - #GTGTGGGC 
480 
- TCCTCACGGC GGAGGCGCTC AGAACCCAAG GTGACTCGCA TGGTGGTGGT AG - #TGGTGCGG 
540 
- CTGTTCGTGG GCTGCTGGCT GCCTTTCTTC ATCGTCAACA TCGTCAACCT GG - #CCTTCACG 
600 
- CTACCCGAGG AGCCCACCTC TGCCGGCCTC TACTTCTTTG TGGTGGTCCT GT - #CTTATGCC 
660 
# 678 CC 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 211 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Rat 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- Val Leu Phe Met Leu Gly Leu Pro - # Phe Leu Ala Thr Gln Asn Ala 
Val 
# 15 
- Ser Tyr Trp Pro Phe Gly Ser Phe - # Leu Cys Arg Leu Val Met Thr 
Leu 
# 30 
- Asp Gly Ile Asn Gln Phe Thr Ser - # Ile Phe Cys Leu Met Val Met 
Ser 
# 45 
- Val Asp Arg Tyr Leu Ala Val Val - # His Pro Leu Arg Ser Ala Arg 
Trp 
# 60 
- Arg Arg Pro Arg Val Ala Lys Leu - # Ala Ser Ala Ala Val Trp Val 
Phe 
# 80 
- Ser Leu Leu Met Ser Leu Pro Leu - # Leu Val Phe Ala Asp Val Gln 
Glu 
# 95 
- Gly Trp Gly Asn Cys Asn Leu Ser - # Trp Pro Glu Pro Val Gly Met 
Trp 
# 110 
- Gly Ala Ala Phe Ile Thr Tyr Thr - # Ser Val Leu Gly Phe Phe Gly 
Pro 
# 125 
- Leu Leu Val Ile Cys Met Cys Tyr - # Leu Leu Ile Val Val Lys Val 
Lys 
# 140 
- Ala Ala Gly Met Arg Val Gly Ser - # Ser Arg Arg Arg Arg Ser Glu 
Pro 
# 160 
- Lys Val Thr Arg Met Val Val Val - # Val Val Arg Leu Phe Val Gly 
Cys 
# 175 
- Trp Leu Pro Phe Phe Ile Val Asn - # Ile Val Asn Leu Ala Phe Thr 
Leu 
# 190 
- Pro Glu Glu Pro Thr Ser Ala Gly - # Leu Tyr Phe Phe Val Val Val 
Leu 
# 205 
- Ser Tyr Ala 
210 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 18 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: YES 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 18 GG 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 18 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 18 CC 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: YES 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
# 20 GAGG 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
# 20 CCTA 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 26 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 4 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 6 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 9 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 19..21 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
# 26 YCNN NGCTGA 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 19..20 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
# 25 CTNN MTGAC 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 26 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 10 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 19 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 23..24 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
# 26 TGNK KRNNTT 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 16 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
# 20 TGCC 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 7 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
# 17 C 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 21 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 6 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 8 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 13..15 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
#21 GYTG G 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 19 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 3 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 6 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 12 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
# 19 AYA 
- (2) INFORMATION FOR SEQ ID NO:16: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 19 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 5 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 8 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 11 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 14 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
# 19 SGG 
- (2) INFORMATION FOR SEQ ID NO:17: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 22 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Synthetic 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 5 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 8 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 12..13 
- (ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- - #base 
(B) LOCATION: 17 
- (x) PUBLICATION INFORMATION: 
(A) AUTHORS: Hadcock Dr - #., John R. 
# Dr. Ozenberger, Bradley A. 
# Dr. Pausch, Mark H. 
(B) TITLE: Receptor Ide - #ntification Method 
(G) DATE: 20-DEC-1996 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
# 22CAG TA 
- (2) INFORMATION FOR SEQ ID NO:18: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 211 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Mouse 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- Glu Leu Phe Met Leu Gly Leu Pro - # Phe Leu Ala Met Gln Val Ala 
Leu 
# 15 
- Val His Trp Pro Phe Gly Lys Ala - # Ile Cys Arg Val Val Met Thr 
Val 
# 30 
- Asp Gly Ile Asn Gln Phe Thr Ser - # Ile Phe Cys Leu Thr Val Met 
Ser 
# 45 
- Ile Asp Arg Tyr Leu Ala Val Val - # His Pro Ile Lys Ser Ala Lys 
Trp 
# 60 
- Arg Arg Pro Arg Thr Ala Lys Met - # Ile Asn Val Ala Val Trp Cys 
Val 
# 80 
- Ser Leu Leu Val Ile Leu Pro Ile - # Met Ile Tyr Ala Gly Leu Arg 
Asn 
# 95 
- Gln Trp Gly Ser Cys Thr Ile Asn - # Trp Pro Gly Glu Ser Gly Ala 
Trp 
# 110 
- Tyr Thr Gly Phe Ile Ile Tyr Ala - # Phe Ile Leu Gly Phe Leu Val 
Pro 
# 125 
- Leu Thr Ile Ile Cys Leu Cys Tyr - # Leu Phe Ile Ile Ile Lys Val 
Lys 
# 140 
- Ser Ser Gly Ile Arg Val Gly Ser - # Ser Lys Arg Lys Lys Ser Glu 
Lys 
# 160 
- Lys Val Thr Arg Met Val Ser Ile - # Val Val Ala Val Phe Ile Phe 
Cys 
# 175 
- Trp Leu Pro Phe Tyr Ile Phe Asn - # Val Ser Ser Val Ser Val Ala 
Ile 
# 190 
- Ser Pro Thr Pro Ala Leu Lys Gly - # Met Phe Asp Phe Val Val Ile 
Leu 
# 205 
- Thr Tyr Ala 
210 
- (2) INFORMATION FOR SEQ ID NO:19: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 211 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Human 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- Glu Leu Leu Met Leu Ser Val Pro - # Phe Leu Val Thr Ser Thr Leu 
Leu 
# 15 
- Arg His Trp Pro Phe Gly Ala Leu - # Leu Cys Arg Leu Val Leu Ser 
Val 
# 30 
- Asp Ala Val Asn Met Phe Thr Ser - # Ile Tyr Cys Leu Thr Val Leu 
Ser 
# 45 
- Val Asp Arg Tyr Val Ala Val Val - # His Pro Ile Lys Ala Ala Arg 
Tyr 
# 60 
- Arg Arg Pro Thr Val Ala Lys Val - # Val Asn Leu Gly Val Trp Val 
Leu 
# 80 
- Ser Leu Leu Val Ile Leu Pro Ile - # Val Val Phe Ala Ala Asn Ser 
Asp 
# 95 
- Gly Thr Val Ala Cys Asn Met Leu - # Met Pro Glu Pro Ala Gln Arg 
Trp 
# 110 
- Leu Val Gly Phe Val Leu Tyr Thr - # Phe Leu Met Gly Phe Ile Leu 
Pro 
# 125 
- Val Gly Ala Ile Cys Leu Cys Tyr - # Val Leu Ile Ile Ala Lys Met 
Arg 
# 140 
- Met Val Ala Leu Lys Ala Gly Trp - # Gln Gln Arg Lys Arg Ser Glu 
Arg 
# 160 
- Lys Ile Thr Leu Met Val Met Met - # Val Val Met Val Phe Val Ile 
Cys 
# 175 
- Trp Met Pro Phe Tyr Val Val Gln - # Leu Val Asn Val Ala Phe Ala 
Glu 
# 190 
- Gln Asp Asp Ala Thr Val Ser Gln - # Leu Tyr Phe Phe Ser Val Ile 
Leu 
# 205 
- Gly Tyr Ala 
210 
__________________________________________________________________________