Polynucleotides that encode the calcitonin gene-related peptide receptor coponent factor (HOUNDC44)

Human CGRP-RCF polypeptides and DNA (RNA) encoding such CGRP-RCF and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such CGRP-RCF for the treatment of diabetes, migrane, pain and inflammation, Parkinson's disease, acute heart failure, hypotension, urinary retention, osteoporosis, hypertension, angina pectoris, myocardial infarction, ulcers, asthma, allergies, psychosis, depression, vomiting, benign prostatic hypertrophy, Paget's disease, obesity, cancer, gigantism and the like. Antagonists against such CGRP-RCF and their use as a therapeutic to treat diabetes, migrane, pain and inflammation, Parkinson's disease, acute heart failure, hypotension, urinary retention, osteoporosis, hypertension, angina pectoris, myocardial infarction, ulcers, asthma, allergies, psychosis, depression, vomiting, benign prostatic hypertrophy, Paget's disease, obesity, cancer, gigantism and the like are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Also disclosed are diagnostic assays for detecting mutations in the polynucleotides encoding the CGRP-RCF and for detecting altered levels of the polypeptide in a host.

FIELD OF THE INVENTION 
This invention relates, in part, to newly identified polynucleotides and 
polypeptides; variants and derivatives of the polynucleotides and 
polypeptides; processes for making the polynucleotides and the 
polypeptides, and their variants and derivatives; agonists and antagonists 
of the polypeptides; and uses of the polynucleotides, polypeptides, 
variants, derivatives, agonists and antagonists. In particular, in these 
and in other regards, the invention relates to polynucleotides and 
polypeptides of human Calcitonin Gene-related Peptide Receptor Component 
Factor, hereinafter referred to as "CGRP-RCF". 
BACKGROUND OF THE INVENTION 
Calcitonin gene related peptide (CGRP) is a 37 amino acid carboxyl-amidated 
neuropeptide secreted by the nerves of the central and peripheral nervous 
systems and exists as highly homologous .alpha. or .beta. isoforms in both 
human and rat (Amara, et al. Nature 298:240-244 (1982); Amara, et al. 
Science 229:1094-1097 (1985)). .alpha.-and .beta.-CGRP display very 
similar biological activities, including peripheral and cerebral 
vasodilation (Brain, et al., Nature 313:54-56 (1985)), cardiac 
acceleration, (Sigrist, et al., Endocrinology 119:381-389 (1986)), 
regulation of calcium metabolism (Grunditz, et al., Endocrinology 
119:2313-2324 (1986)), reduction of intestinal motility (Fargeas, et al., 
Peptides 6:1167-1171(1985)), regulation of glucose metabolism (reduction 
of insulin secretion and insulin sensitivity) (Hermansen, et al., Reg. 
Peptides 27:149-157) (1990)), diminution of appetite (Molina, et al., 
Diabetes 39:260-265 (1990)) and reduction of growth hormone release 
(Tannenbaum, et al., Endocrinology 116:2685-2687 (1985)). The two CGRP 
peptides differ by three amino acids in humans and one amino acid in rats. 
The amino acid sequences of CGRP peptides are well conserved among species 
and can be considered as members of a family of peptides including the 
related peptides amylin (46% homology), salmon calcitonin (32% homology), 
and adrenomedullin (24% homology). These peptides in general have 
N-terminal ring structures of 6-7 amino acids involving a disulfide bridge 
and an amidated C-terminal end (Muff, et al., Eur J Endocrinol. 133:17-20 
(1995); Goodman, et al., Life Sci. 38:2169-2178 (1986); Poyner, D. R. 
Pharmac. Ther. 56:23-51 (1992)). 
CGRP peptides are localized predominantly in sensory afferent nerves and 
central neurons (Goodman, et al., Life Sci. 38:2169-2178 (1986); Poyner, 
D. R. Pharmac. Ther. 56:23-51 (1992)). When released from the cell, the 
peptides initiate their biological responses by binding to specific cell 
surface receptors which are predominantly coupled to the activation of 
adenylyl cyclase (Brain, et al., Nature 313:54-56 (1985); Kubota, et al, 
Biochem Biophys Res Commun 132:88-94 (1985)). CGRP receptors have been 
identified and pharmacologically evaluated in several tissues and cells, 
including brain, cardiovascular, endothelial and smooth muscle (Poyner, D. 
R. Pharmac. Ther. 56:23-51 (1992)). Multiple CGRP receptors have been 
observed, based on pharmacological properties. These receptors they are 
divided into at least two subtypes and denoted as CGRP.sub.1 and 
CGRP.sub.2, according to the classification of Dennis and colleagues 
(Dennis, et al., Brain Res. 539:59-66 (1991)). CGRP (Molina, et al. 
Diabetes 39:260-265 (1990); Tannenbaum, et al., Endocrinology 
116:2685-2687 (1985); Muff, et al., Eur J Endocrinol. 133: 17-20( 1995); 
Goodman, et al., Life Sci. 38:2 169-2178 (1986); Poyner, D. R., Pharmac. 
Ther. 56:23-51 (1992); Kubota, et al., Biochem Biophys Res Commun 
132:88-94 (1985); Dennis, et al., Brain Res. 539:59-66 (1991); Adams, et 
al., Science 252:1651-1656 (1991); Adams, et al., Nature 355:632-634 
(1992); Adams, et al., Nature 377:3-174 (1995); Chang, et al., Neuron 
11:1187-1195 (1993); Fluhmann, et al., Biochem. Biophy. Res. Comm. 
206:341-347 (1995); Jelinek, et al., Science 259:1614-1616 (1993); Kozak, 
M., Proc. Natl. Acad. Sci. (USA), 92:2662-2666 (1995); Aiyar, et al., Mol. 
Cell. Bio. 131:75-86 (1994); Sambrook, et al., Molecular Cloning. Cold 
Spring Harbor Laboratory Press (1989); Nuovo, J. G., PCR in situ 
Hybridization: protocols and applications. Raven Press, New York (1992); 
Aiyar, et al. Endocrinology 129:965-969 (1991); Lin, et al., Science 
254:1022-1024 (1991); Hirata, et al., Biochem. Biophy. Res. Commun. 
151:1113-1121 (1988); Nawa, et al., Nature 312:729-734 (1984); Kozak, M. 
J., Mol. Biol. 196:947-950 (1987); Dohlman, et al., Annu. Rev. Biochem. 
60:653-688 (1991); Jackson, T., Pharmocol. Ther. 50:425-442 (1991); 
Ishihara, et al., EMBO J., 10:1635-1641 (1991); Juppner, et al., Science 
254:1024-1026 (1991); Thorens, B., Proc. Natl. Acad. Sci. U.S.A., 
89:8641-8645 (1992); van Valen, et al., Neurosci. Lett. 119:195-198 
(1990); Stangl, et al., Endocrinology 132:744-750 (1993); Kitamura, et 
al., FEBS Lett., 338:306-310 (1994)), which lacks 7 N-terminal amino acid 
residues, is a selective antagonist of CGRP.sub.1 receptors, whereas the 
linear analog of CGRP, diacetoamidomethyl cysteine CGRP 
({Cys(ACM)2,7}CGRP), is a selective agonist of CGRP.sub.2 receptors 
(Dennis, et al., Brain Res. 539:59-66 (1991)). 
As indicated above, CGRP has a plethora of functions in the body and is 
known to be the most potent vasodilator and neuromodulator. Although we 
recently reported the cDNA encoding the human CGRP-type I receptor (Aiyar 
et al., Journal of Biological Chemistry 271:11325-11329 (1996)), we 
observed during our characterization studies that the receptor did not 
confer CGRP responsiveness in Xenopus oocytes, in transiently expressing 
COS cells, and in stably expressing Baculovirus and Drosophila cells. In 
contrast to these observations, we are able to confer reasonable levels of 
CGRP responsiveness in stably transfected human embyonic kidney 293 cells. 
These observations suggest the requirement of an additional human 
complementary factor for functional coupling of the CGRP receptor in all 
of these heterologous systems. 
Recently, Luebke et al., (Proc. Natl. Acad. Science, 93:3455-3460 (1996)) 
used an expression cloning strategy to isolate a guinea pig cDNA that 
encodes a protein (CGRP-receptor component protein) that confers CGRP 
responsiveness in oocytes. Utilizing this information and using the 
expressed sequence tag (EST) analysis (Adams, et al., Science 
252:1651-1656 (1991); Adams, et al., Nature 355:632-634 (1992); Adams, et 
al., Nature 377:3-174 (1995)) we identified the human homologue of the 
guinea pig CGRP-RCP. A full length cDNA clone is identified from a human 
adipocytes osteoclastoma cDNA library. 
Although numerous groups have indicated the absolute requirement for 
complementary RCFs for functional signaling in heterologous systems for 
several 7TM receptors (JBC 266:12560, 1991 and FEBS Lett. 291:208, 1991), 
none were able to successfully isolate the cDNA encoding these specific 
factors. 
The identification and isolation of a complementary human factor neccessary 
for expression of the CGRP type I receptor in heterologous systems, 
provides an opportunity to delineate more exactly or enhance the signaling 
pathways that are activated by the receptor. This also offers a novel 
approach to identify functionally the increased number of orphan cDNAs 
encoding suspected G-protein linked receptors whose ligands are still 
unknown. 
Clearly, there is a need for factors necessary for correct signal 
transduction of CGRP receptors (or even other G-protein coupled receptors) 
and their roles in dysfunction and disease. There is a need, therefore, 
for identification and characterization of such factors which can play a 
role in preventing, ameliorating or correcting dysfunctions or diseases, 
such as, but not limited to, diabetes, migrane, pain and inflammation. 
SUMMARY OF THE INVENTION 
Toward these ends, and others, it is an object of the present invention to 
provide polypeptides, inter alia, that have been identified as novel 
CGRP-RCF by homology between the amino acid sequence set out in FIG. 1 and 
known amino acid sequences of other proteins such as guinea pig CGRP-RCP 
described in by Luebke in the Proc. Natl. Acad. Sci. 93:3455-3460 (1996). 
It is a further object of the invention, moreover, to provide 
polynucleotides that encode CGRP-RCF, particularly polynucleotides that 
encode the polypeptide herein designated CGRP-RCF. 
In a particularly preferred embodiment of this aspect of the invention the 
polynucleotide comprises the region encoding human CGRP-RCF in the 
sequence set out in FIG. 1. 
In accordance with this aspect of the present invention there is provided 
an isolated nucleic acid molecule encoding a mature polypeptide expressed 
by the human cDNA contained in ATCC Deposit No. 98105. 
In accordance with this aspect of the invention there are provided isolated 
nucleic acid molecules encoding human CGRP-RCF, including mRNAs, cDNAs, 
genomic DNAs and, in further embodiments of this aspect of the invention, 
biologically, diagnostically, clinically or therapeutically useful 
variants, analogs or derivatives thereof, or fragments thereof, including 
fragments of the variants, analogs and derivatives. 
Among the particularly preferred embodiments of this aspect of the 
invention are naturally occurring allelic variants of human CGRP-RCF. 
It also is an object of the invention to provide CGRP-RCF polypeptides, 
particularly human CGRP-RCF polypeptides, that may be employed for 
therapeutic purposes, for example, to treat diabetes, migrane, pain and 
inflammation, Parkinson's disease, acute heart failure, hypotension, 
urinary retention, osteoporosis, hypertension, angina pectoris, myocardial 
infarction, ulcers, asthma, allergies, psychosis, depression, vomiting, 
benign prostatic hypertrophy, Paget's disease, obesity, cancer, gigantism 
and the like. 
In accordance with this aspect of the invention there are provided novel 
polypeptides of human origin referred to herein as CGRP-RCF as well as 
biologically, diagnostically or therapeutically useful fragments, variants 
and derivatives thereof, variants and derivatives of the fragments, and 
analogs of the foregoing. 
Among the particularly preferred embodiments of this aspect of the 
invention are variants of human CGRP-RCF encoded by naturally occurring 
alleles of the human CGRP-RCF gene. 
In accordance with another aspect of the present invention there are 
provided methods of screening for compounds which bind to and activate or 
inhibit activation of the CGRP receptor or CGRP/CGRP-RCF receptor 
polypeptides and for receptor ligands. 
It is another object of the invention to provide a process for producing 
the aforementioned polypeptides, polypeptide fragments, variants and 
derivatives, fragments of the variants and derivatives, and analogs of the 
foregoing. 
In a preferred embodiment of this aspect of the invention there are 
provided methods for producing the aforementioned CGRP-RCF polypeptides 
comprising culturing host cells having expressibly incorporated therein an 
exogenously-derived human CGRP-RCF-encoding polynucleotide under 
conditions for expression of human CGRP-RCF in the host and then 
recovering the expressed polypeptide. 
In accordance with another object the invention there are provided 
products, compositions, processes and methods that utilize the 
aforementioned polypeptides and polynucleotides for research, biological, 
clinical and therapeutic purposes, inter alia. 
In accordance with certain preferred embodiments of this aspect of the 
invention, there are provided products, compositions and methods, inter 
alia, for, among other things: assessing CGRP-RCF expression in cells by 
determining CGRP-RCF polypeptides or CGRP-RCF-encoding mRNA; treating 
diabetes, migrane, pain and inflammation, Parkinson's disease, acute heart 
failure, hypotension, urinary retention, osteoporosis, hypertension, 
angina pectoris, myocardial infarction, ulcers, asthma, allergies, 
psychosis, depression, vomiting, benign prostatic hypertrophy, Paget's 
disease, obesity, cancer, gigantism and the like, in vitro, ex vivo or in 
vivo by exposing cells to CGRP-RCF polypeptides or polynucleotides as 
disclosed herein; assaying genetic variation and aberrations, such as 
defects, in CGRP-RCF genes; and administering a CGRP-RCF polypeptide or 
polynucleotide to an organism to augment CGRP-RCF function or remediate 
CGRP-RCF dysfunction. 
In accordance with still another embodiment of the present invention there 
is provided a process of using such activating compounds to stimulate the 
CGRP receptor polypeptide for the treatment of conditions related to the 
under-expression of the CGRP-RCF. 
In accordance with another aspect of the present invention there is 
provided a process of using such inhibiting compounds for treating 
conditions associated with over-expression of the CGRP-RCF. 
In accordance with yet another aspect of the present invention there is 
provided non-naturally occurring synthetic, isolated and/or recombinant 
CGRP-RCF polypeptides which are fragments, consensus fragments and/or 
sequences having conservative amino acid substitutions, of at least one 
domain of the CGRP-RCF of the present invention, such that the CGRP-RCF 
may bind to CGRP receptor ligands or to CGRP receptor, or which may also 
modulate, quantitatively or qualitatively, CGRP receptor ligand binding. 
In accordance with still another aspect of the present invention there are 
provided synthetic or recombinant CGRP-RCF polypeptides, conservative 
substitution and derivatives thereof, antibodies, anti-idiotype 
antibodies, compositions and methods that can be useful as potential 
modulators of CGRP-RCF function, by binding to CGRP receptor ligands or 
CGRP receptor or modulating ligand binding, which may be used in 
diagnostic, therapeutic and/or research applications. 
It is still another object of the present invention to provide synthetic, 
isolated or recombinant polypeptides which are designed to inhibit or 
mimic various CGRP-RCF or fragments thereof. 
In accordance with certain preferred embodiments of this and other aspects 
of the invention there are provided probes that hybridize to human 
CGRP-RCF sequences. 
In certain additional preferred embodiments of this aspect of the invention 
there are provided antibodies against CGRP-RCF polypeptides. In certain 
particularly preferred embodiments in this regard, the antibodies are 
highly selective for human CGRP-RCF. 
In accordance with another aspect of the present invention, there are 
provided CGRP-RCF and CGRP-RCF/CGRP receptor agonists. Among preferred 
agonists are molecules that mimic CGRP-RCF, that bind to CGRP receptor 
molecules or CGRP-RCF, and that elicit or augment CGRP-RCF-induced 
responses. Also among preferred agonists are molecules that interact with 
CGRP-RCF or CGRP-RCF/CGRP-RCF receptor, or with other modulators of 
CGRP-RCF activities, and thereby potentiate or augment an effect of 
CGRP-RCF or more than one effect of CGRP-RCF. 
In accordance with yet another aspect of the present invention, there are 
provided CGRP-RCF and CGRP-RCF/CGRP receptor antagonists. Among preferred 
antagonists are those which mimic CGRP-RCF so as to bind to CGRP receptor 
or CGRP-RCF but inhibit CGRP-RCF-induced response or more than one 
CGRP-RCF-induced response. Also among preferred antagonists are molecules 
that bind to or interact with CGRP-RCF or CGRP/CGRP-RCF receptor so as to 
inhibit an effect of CGRP-RCF or more than one effect of CGRP-RCF or which 
prevent expression of CGRP-RCF. 
In a further aspect of the invention there are provided compositions 
comprising a CGRP-RCF polynucleotide or a CGRP-RCF polypeptide for 
administration to cells in vitro, to cells ex vivo and to cells in vivo, 
or to a multicellular organism. In certain particularly preferred 
embodiments of this aspect of the invention, the compositions comprise a 
CGRP-RCF polynucleotide for expression of a CGRP-RCF polypeptide in a host 
organism for treatment of disease. Particularly preferred in this regard 
is expression in a human patient for treatment of a dysfunction associated 
with aberrant endogenous activity of CGRP-RCF. 
Other objects, features, advantages and aspects of the present invention 
will become apparent to those of skill in the art from the following 
description. It should be understood, however, that the following 
description and the specific examples, while indicating preferred 
embodiments of the invention, are given by way of illustration only. 
Various changes and modifications within the spirit and scope of the 
disclosed invention will become readily apparent to those skilled in the 
art from reading the following description and from reading the other 
parts of the present disclosure.

GLOSSARY 
The following illustrative explanations are provided to facilitate 
understanding of certain terms used frequently herein, particularly in the 
examples. The explanations are provided as a convenience and are not 
limitative of the invention. 
DIGESTION of DNA refers to catalytic cleavage of the DNA with a restriction 
enzyme that acts only at certain sequences in the DNA. The various 
restriction enzymes referred to herein are commercially available and 
their reaction conditions, cofactors and other requirements for use are 
known and routine to the skilled artisan. 
For analytical purposes, typically, 1 .mu.g of plasmid or DNA fragment is 
digested with about 2 units of enzyme in about 20 .mu.l of reaction 
buffer. For the purpose of isolating DNA fragments for plasmid 
construction, typically 5 to 50 .mu.g of DNA are digested with 20 to 250 
units of enzyme in proportionately larger volumes. 
Appropriate buffers and substrate amounts for particular restriction 
enzymes are described in standard laboratory manuals, such as those 
referenced below, and they are specified by commercial suppliers. 
Incubation times of about 1 hour at 37.degree. C. are ordinarily used, but 
conditions may vary in accordance with standard procedures, the supplier's 
instructions and the particulars of the reaction. After digestion, 
reactions may be analyzed, and fragments may be purified by 
electrophoresis through an agarose or polyacrylamide gel, using well known 
methods that are routine for those skilled in the art. 
GENETIC ELEMENT generally means a polynucleotide comprising a region that 
encodes a polypeptide or a region that regulates transcription or 
translation or other processes important to expression of the polypeptide 
in a host cell, or a polynucleotide comprising both a region that encodes 
a polypeptide and a region operably linked thereto that regulates 
expression. 
Genetic elements may be comprised within a vector that replicates as an 
episomal element; that is, as a molecule physically independent of the 
host cell genome. They may be comprised within mini-chromosomes, such as 
those that arise during amplification of transfected DNA by methotrexate 
selection in eukaryotic cells. Genetic elements also may be comprised 
within a host cell genome; not in their natural state but, rather, 
following manipulation such as isolation, cloning and introduction into a 
host cell in the form of purified DNA or in a vector, among others. 
ISOLATED means altered "by the hand of man" from its natural state; i.e., 
that, if it occurs in nature, it has been changed or removed from its 
original environment, or both. 
For example, a naturally occurring polynucleotide or a polypeptide 
naturally present in a living animal in its natural state is not 
"isolated," but the same polynucleotide or polypeptide separated from the 
coexisting materials of its natural state is "isolated", as the term is 
employed herein. For example, with respect to polynucleotides, the term 
isolated means that it is separated from the chromosome and cell in which 
it naturally occurs. 
As part of or following isolation, such polynucleotides can be joined to 
other polynucleotides, such as DNAs, for mutagenesis, to form fusion 
proteins, and for propagation or expression in a host, for instance. The 
isolated polynucleotides, alone or joined to other polynucleotides such as 
vectors, can be introduced into host cells, in culture or in whole 
organisms. Introduced into host cells in culture or in whole organisms, 
such DNAs still would be isolated, as the term is used herein, because 
they would not be in their naturally occurring form or environment. 
Similarly, the polynucleotides and polypeptides may occur in a 
composition, such as a media formulations, solutions for introduction of 
polynucleotides or polypeptides, for example, into cells, compositions or 
solutions for chemical or enzymatic reactions, for instance, which are not 
naturally occurring compositions, and, therein remain isolated 
polynucleotides or polypeptides within the meaning of that term as it is 
employed herein. 
LIGATION refers to the process of forming phosphodiester bonds between two 
or more polynucleotides, which most often are double stranded DNAs. 
Techniques for ligation are well known to the art and protocols for 
ligation are described in standard laboratory manuals and references, such 
as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 
(1989) and Maniatis et al., pg. 146, as cited below. 
OLIGONUCLEOTIDE(S) refers to relatively short polynucleotides. Often the 
term refers to single-stranded deoxyribonucleotides, but it can refer as 
well to single-or double-stranded ribonucleotides, RNA:DNA hybrids and 
double-stranded DNAs, among others. 
Oligonucleotides, such as single-stranded DNA probe oligonucleotides, often 
are synthesized by chemical methods, such as those implemented on 
automated oligonucleotide synthesizers. However, oligonucleotides can be 
made by a variety of other methods, including in vitro recombinant 
DNA-mediated techniques and by expression of DNAs in cells and organisms. 
Initially, chemically synthesized DNAs typically are obtained without a 5' 
phosphate. The 5' ends of such oligonucleotides are not substrates for 
phosphodiester bond formation by ligation reactions that employ DNA 
ligases typically used to form recombinant DNA molecules. Where ligation 
of such oligonucleotides is desired, a phosphate can be added by standard 
techniques, such as those that employ a kinase and ATP. 
The 3' end of a chemically synthesized oligonucleotide generally has a free 
hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, 
readily will form a phosphodiester bond with a 5' phosphate of another 
polynucleotide, such as another oligonucleotide. As is well known, this 
reaction can be prevented selectively, where desired, by removing the 5' 
phosphates of the other polynucleotide(s) prior to ligation. 
PLASMIDS generally are designated herein by a lower case p preceded and/or 
followed by capital letters and/or numbers, in accordance with standard 
naming conventions that are familiar to those of skill in the art. 
Starting plasmids disclosed herein are either commercially available, 
publicly available on an unrestricted basis, or can be constructed from 
available plasmids by routine application of well known, published 
procedures. Many plasmids and other cloning and expression vectors that 
can be used in accordance with the present invention are well known and 
readily available to those of skill in the art. Moreover, those of skill 
readily may construct any number of other plasmids suitable for use in the 
invention. The properties, construction and use of such plasmids, as well 
as other vectors, in the present invention will be readily apparent to 
those of skill from the present disclosure. 
POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide or 
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA 
or DNA. Thus, for instance, polynucleotides as used herein refers to, 
among others, single-and double-stranded DNA, DNA that is a mixture of 
single-and double-stranded regions, single- and double-stranded RNA, and 
RNA that is mixture of single- and double-stranded regions, hybrid 
molecules comprising DNA and RNA that may be single-stranded or, more 
typically, double-stranded or a mixture of single- and double-stranded 
regions. 
In addition, polynucleotide as used herein refers to triple-stranded 
regions comprising RNA or DNA or both RNA and DNA. The strands in such 
regions may be from the same molecule or from different molecules. The 
regions may include all of one or more of the molecules, but more 
typically involve only a region of some of the molecules. One of the 
molecules of a triple-helical region often is an oligonucleotide. 
As used herein, the term polynucleotide includes DNAs or RNAs as described 
above that contain one or more modified bases. Thus, DNAs or RNAs with 
backbones modified for stability or for other reasons are 
"polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs 
comprising unusual bases, such as inosine, or modified bases, such as 
tritylated bases, to name just two examples, are polynucleotides as the 
term is used herein. 
It will be appreciated that a great variety of modifications have been made 
to DNA and RNA that serve many useful purposes known to those of skill in 
the art. The term polynucleotide as it is employed herein embraces such 
chemically, enzymatically or metabolically modified forms of 
polynucleotides, as well as the chemical forms of DNA and RNA 
characteristic of viruses and cells, including simple and complex cells, 
inter alia. 
POLYPEPTIDES, as used herein, includes all polypeptides as described below. 
The basic structure of polypeptides is well known and has been described 
in innumerable textbooks and other publications in the art. In this 
context, the term is used herein to refer to any peptide or protein 
comprising two or more amino acids joined to each other in a linear chain 
by peptide bonds. As used herein, the term refers to both short chains, 
which also commonly are referred to in the art as peptides, oligopeptides 
and oligomers, for example, and to longer chains, which generally are 
referred to in the art as proteins, of which there are many types. 
It will be appreciated that polypeptides often contain amino acids other 
than the 20 amino acids commonly referred to as the 20 naturally occurring 
amino acids, and that many amino acids, including the terminal amino 
acids, may be modified in a given polypeptide, either by natural 
processes, such as processing and other posttranslational modifications, 
but also by chemical modification techniques which are well known to the 
art. Even the common modifications that occur naturally in polypeptides 
are too numerous to list exhaustively here, but they are well described in 
basic texts and in more detailed monographs, as well as in a voluminous 
research literature, and they are well known to those of skill in the art. 
Among the known modifications which may be present in polypeptides of the 
present are, to name an illustrative few, acetylation, acylation, 
ADP-ribosylation, amidation, covalent attachment of flavin, covalent 
attachment of a heme moiety, covalent attachment of a nucleotide or 
nucleotide derivative, covalent attachment of a lipid or lipid derivative, 
covalent attachment of phosphotidylinositol, cross-linking, cyclization, 
disulfide bond formation, demethylation, formation of covalent 
cross-links, formation of cystine, formation of pyroglutamate, 
formylation, gamma-carboxylation, glycosylation, GPI anchor formation, 
hydroxylation, iodination, methylation, myristoylation, oxidation, 
proteolytic processing, phosphorylation, prenylation, racemization, 
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to 
proteins such as arginylation, and ubiquitination. 
Such modifications are well known to those of skill and have been described 
in great detail in the scientific literature. Several particularly common 
modifications, glycosylation, lipid attachment, sulfation, 
gamma-carboxylation of glutamic acid residues, hydroxylation and 
ADP-ribosylation, for instance, are described in most basic texts, such 
as, for instance PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. 
E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed 
reviews are available on this subject, such as, for example, those 
provided by Wold, F., Posttranslational Protein Modifications: 
Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT 
MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York 
(1983); Seifter et al., Analysis for protein modifications and nonprotein 
cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Protein 
Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 
663:48-62 (1992). 
It will be appreciated, as is well known and as noted above, that 
polypeptides are not always entirely linear. For instance, polypeptides 
may be branched as a result of ubiquitination, and they may be circular, 
with or without branching, generally as a result of posttranslation 
events, including natural processing event and events brought about by 
human manipulation which do not occur naturally. Circular, branched and 
branched circular polypeptides may be synthesized by non-translation 
natural process and by entirely synthetic methods, as well. 
Modifications can occur anywhere in a polypeptide, including the peptide 
backbone, the amino acid side-chains and the amino or carboxyl termini. In 
fact, blockage of the amino or carboxyl group in a polypeptide, or both, 
by a covalent modification, is common in naturally occurring and synthetic 
polypeptides and such modifications may be present in polypeptides of the 
present invention, as well. For instance, the amino terminal residue of 
polypeptides made in E. coli, prior to proteolytic processing, almost 
invariably will be N-formylmethionine. 
The modifications that occur in a polypeptide often will be a function of 
how it is made. For polypeptides made by expressing a cloned gene in a 
host, for instance, the nature and extent of the modifications in large 
part will be determined by the host cell posttranslational modification 
capacity and the modification signals present in the polypeptide amino 
acid sequence. For instance, as is well known, glycosylation often does 
not occur in bacterial hosts such as E. coli. Accordingly, when 
glycosylation is desired, a polypeptide should be expressed in a 
glycosylating host, generally a eukaryotic cell. Insect cell often carry 
out the same posttranslational glycosylations as mammalian cells and, for 
this reason, insect cell expression systems have been developed to express 
efficiently mammalian proteins having native patterns of glycosylation, 
inter alia. Similar considerations apply to other modifications. 
It will be appreciated that the same type of modification may be present in 
the same or varying degree at several sites in a given polypeptide. Also, 
a given polypeptide may contain many types of modifications. 
In general, as used herein, the term polypeptide encompasses all such 
modifications, particularly those that are present in polypeptides 
synthesized by expressing a polynucleotide in a host cell. 
VARIANT(S) of polynucleotides or polypeptides, as the term is used herein, 
are polynucleotides or polypeptides that differ from a reference 
polynucleotide or polypeptide, respectively. Variants in this sense are 
described below and elsewhere in the present disclosure in greater detail. 
(1) A polynucleotide that differs in nucleotide sequence from another, 
reference polynucleotide. Generally, differences are limited so that the 
nucleotide sequences of the reference and the variant are closely similar 
overall and, in many regions, identical. 
As noted below, changes in the nucleotide sequence of the variant may be 
silent. That is, they may not alter the amino acids encoded by the 
polynucleotide. Where alterations are limited to silent changes of this 
type a variant will encode a polypeptide with the same amino acid sequence 
as the reference. Also as noted below, changes in the nucleotide sequence 
of the variant may alter the amino acid sequence of a polypeptide encoded 
by the reference polynucleotide. Such nucleotide changes may result in 
amino acid substitutions, additions, deletions, fusions and truncations in 
the polypeptide encoded by the reference sequence, as discussed below. 
(2) A polypeptide that differs in amino acid sequence from another, 
reference polypeptide. Generally, differences are limited so that the 
sequences of the reference and the variant are closely similar overall 
and, in many region, identical. 
A variant and reference polypeptide may differ in amino acid sequence by 
one or more substitutions, additions, deletions, fusions and truncations, 
which may be present in any combination. 
FUSION PROTEINS: EP-A-O 464 533 (Canadian counterpart 2045869) discloses 
fusion proteins comprising various portions of constant region of 
immunoglobin molecules together with another human protein or part 
thereof. In many cases, the Fc part in fusion protein is thoroghly 
advantageous for use in therapy and diagnosis and thus results, for 
example, in improved pharmacokinetic properties (EP-A 0232 262). On the 
other hand, for some uses it would be desirable to be able to delete the 
Fc part after the fusion protein has been expressed, detected and purified 
in the advantageous manner described. This is the case when Fc portion 
proves to be a hindrance to use in therapy and diagnosis, for example when 
the fusion protein is to be used as antigen for immunizations. In drug 
discovery, for example, human proteins, such as, shIL5-.alpha. has been 
fused with Fc portions for the purpose of high-throughput screening assays 
to identify antagonists of hIL-5. See, Bennett et al., Journal of 
Molecular Recognition, 8:52-58 (1995) and Johanson et al., The Journal of 
Biological Chemistry, 270, No. 16, 9459-9471 (1995). 
Thus, this invention also relates to genetically engineered soluble fusion 
proteins comprised from CGRP-RCF, or a portion thereof, and of various 
portions of the constant regions of heavy or light chains of 
immunoglobulins of various subclass (IgG, IgM, IgA, IgE). Preferred as 
immunoglobulin is the constant part of the heavy chain of human IgG, 
particularly IgG 1, where fusion takes place at the hinge region. In a 
particular embodiment, the Fc part can be removed in a simple way by a 
cleavage sequence which is also incorporated and can be cleaved with 
factor Xa. Furthermore, this invention relates to processes for the 
preparation of these fusion by genetic engineering, and to the use thereof 
for diagnosis and therapy. An yet further aspect of the invention also 
relates to polynucleotide encoding such fusion proteins. 
BINDING MOLECULES (or otherwise called INTERACTION MOLECULES or RECEPTOR 
COMPONENT FACTORS) refer to molecules other than CGRP receptor ligands or 
CGRP receptor that specifically bind to or interact with polypeptides of 
the present invention. Such binding molecules are a part of the present 
invention. BINDING MOLECULES also may be non-naturally occurring, such as 
antibodies and antibody-derived reagents that bind specifically to 
polypeptides of the invention. 
CGRP-RCF/CGRP RECEPTOR SYSTEM or CGRP-RCF/CGRP RECEPTOR or simply RECEPTOR 
SYSTEM as used herein refers to a complex formed between CGRP receptor and 
CGRP-RCF. 
DESCRIPTION OF THE INVENTION 
The present invention relates to novel CGRP-RCF polypeptides and 
polynucleotides, among other things, as described in greater detail below. 
In particular, the invention relates to polypeptides and polynucleotides 
of a novel human CGRP-RCF, which is related by amino acid sequence 
homology to guinea pig CGRP-RCP polypeptide. The invention relates 
especially to CGRP-RCF having the nucleotide and amino acid sequences set 
out in FIG. 1, and to the CGRP-RCF nucleotide and amino acid sequences of 
the human cDNA in ATCC Deposit No. 98105, which is herein referred to as 
"the deposited clone" or as the "cDNA of the deposited clone." It will be 
appreciated that the nucleotide and amino acid sequences set out in FIG. 1 
are obtained by sequencing the cDNA of the deposited clone. Hence, the 
sequence of the deposited clone is controlling as to any discrepancies 
between the two and any reference to the sequences of FIG. 1 include 
reference to the sequence of the human cDNA of the deposited clone. 
Polynucleotides 
In accordance with one aspect of the present invention, there are provided 
isolated polynucleotides which encode the CGRP-RCF polypeptide having the 
deduced amino acid sequence of FIG. 1. 
Using the information provided herein, such as the polynucleotide sequence 
set out in FIG. 1, a polynucleotide of the present invention encoding 
human CGRP-RCF polypeptide may be obtained using standard cloning and 
screening procedures, such as those for cloning cDNAs using mRNA from 
human adipocytes from osteoclastoma as starting material. Illustrative of 
the invention, the polynucleotide set out in FIG. 1 is discovered in a 
cDNA library derived from human adipocytes from osteoclastoma using the 
expressed sequence tag (EST) analysis (Adams, et al., Science 
252:1651-1656 (1991); Adams, et al., Nature 355:632-634 (1992); Adams, et 
al., Nature 377 Supp, 3-174) (1995)). 
Human CGRP-RCF of the invention is structurally related to the guinea pig 
CGRP-RCP, as shown by the results of sequencing the cDNA encoding human 
CGRP-RCF in the deposited clone. The cDNA sequence thus obtained is set 
out in FIG. 1. SEQ ID NO: 1. It contains an open reading frame encoding a 
protein of 148 amino acid residues with a deduced molecular weight of 
about 17.7 kDa. CGRP-RCF of FIG. 1 has about 88% identity and about 94% 
similarity with the amino acid sequence of guinea pig CGRP-RCP. 
Polynucleotides of the present invention may be in the form of RNA, such as 
mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA 
obtained by cloning or produced by chemical synthetic techniques or by a 
combination thereof. The DNA may be double-stranded or single-stranded. 
Single-stranded DNA may be the coding strand, also known as the sense 
strand, or it may be the non-coding strand, also referred to as the 
anti-sense strand. 
The coding sequence which encodes the polypeptide may be identical to the 
coding sequence of the polynucleotide shown in FIG. 1. SEQ ID NO: 1. It 
also may be a polynucleotide with a different sequence, which, as a result 
of the redundancy (degeneracy) of the genetic code, encodes the 
polypeptide of the DNA of FIG. 1. 
Polynucleotides of the present invention which encode the polypeptide of 
FIG. 1 may include, but are not limited to the coding sequence for the 
mature polypeptide, by itself; the coding sequence for the mature 
polypeptide and additional coding sequences, such as those encoding a 
leader or secretory sequence, such as a pre-, or pro- or prepro-protein 
sequence; the coding sequence of the mature polypeptide, with or without 
the aforementioned additional coding sequences, together with additional, 
non-coding sequences, including for example, but not limited to introns 
and non-coding 5' and 3' sequences, such as the transcribed, 
non-translated sequences that play a role in transcription, mRNA 
processing--including splicing and polyadenylation signals, for 
example--ribosome binding and stability of mRNA; additional coding 
sequence which codes for additional amino acids, such as those which 
provide additional functionalities. Thus, for instance, the polypeptide 
may be fused to a marker sequence, such as a peptide, which facilitates 
purification of the fused polypeptide. In certain preferred embodiments of 
this aspect of the invention, the marker sequence is a hexa-histidine 
peptide, such as the tag provided in the pQE vector (Qiagen, Inc.), among 
others, many of which are commercially available. As described in Gentz et 
al., Proc. Natl. Acad. Sci., USA 86:821-824 (1989), for instance, 
hexa-histidine provides for convenient purification of the fusion protein. 
The HA tag corresponds to an epitope derived of influenza hemagglutinin 
protein, which has been described by Wilson et al., Cell 37: 767 (1984), 
for instance. 
In accordance with the foregoing, the term "polynucleotide encoding a 
polypeptide" as used herein encompasses polynucleotides which include a 
sequence encoding a polypeptide of the present invention, particularly the 
human CGRP-RCF having the amino acid sequence set out in FIG. 1. The term 
also encompasses polynucleotides that include a single continuous region 
or discontinuous regions encoding the polypeptide (for example, 
interrupted by introns) together with additional regions, that also may 
contain coding and/or non-coding sequences. 
The present invention further relates to variants of the herein above 
described polynucleotides which encode for fragments, analogs and 
derivatives of the polypeptide having the deduced amino acid sequence of 
FIG. 1. A variant of the polynucleotide may be a naturally occurring 
variant such as a naturally occurring allelic variant, or it may be a 
variant that is not known to occur naturally. Such non-naturally occurring 
variants of the polynucleotide may be made by mutagenesis techniques, 
including those applied to polynucleotides, cells or organisms. 
Among variants in this regard are variants that differ from the 
aforementioned polynucleotides by nucleotide substitutions, deletions or 
additions. The substitutions, deletions or additions may involve one or 
more nucleotides. The variants may be altered in coding or non-coding 
regions or both. Alterations in the coding regions may produce 
conservative or non-conservative amino acid substitutions, deletions or 
additions. 
Among the particularly preferred embodiments of the invention in this 
regard are polynucleotides encoding polypeptides having the amino acid 
sequence of CGRP-RCF set out in FIG. 1; variants, analogs, derivatives and 
fragments thereof, and fragments of the variants, analogs and derivatives. 
Further particularly preferred in this regard are polynucleotides encoding 
CGRP-RCF variants, analogs, derivatives and fragments, and variants, 
analogs and derivatives of the fragments, which have the amino acid 
sequence of the CGRP-RCF polypeptide of FIG. 1 in which several, a few, 5 
to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, 
deleted or added, in any combination. Especially preferred among these are 
silent substitutions, additions and deletions, which do not alter the 
properties and activities of the CGRP-RCF. Also especially preferred in 
this regard are conservative substitutions. Most highly preferred are 
polynucleotides encoding polypeptides having the amino acid sequence of 
FIG. 1, without substitutions. 
Further preferred embodiments of the invention are polynucleotides that are 
at least 70% identical to a polynucleotide encoding the CGRP-RCF 
polypeptide having the amino acid sequence set out in FIG. 1, and 
polynucleotides which are complementary to such polynucleotides. 
Alternatively, most highly preferred are polynucleotides that comprise a 
region that is at least 80% identical to a polynucleotide encoding the 
CGRP-RCF polypeptide of the human cDNA of the deposited clone and 
polynucleotides complementary thereto. In this regard, polynucleotides at 
least 90% identical to the same are particularly preferred, and among 
these particularly preferred polynucleotides, those with at least 95% are 
especially preferred. Furthermore, those with at least 97% are highly 
preferred among those with at least 95%, and among these those with at 
least 98% and at least 99% are particularly highly preferred, with at 
least 99% being the more preferred. 
Particularly preferred embodiments in this respect, moreover, are 
polynucleotides which encode polypeptides which retain substantially the 
same biological function or activity as the mature polypeptide encoded by 
the cDNA of FIG. 1. 
The present invention further relates to polynucleotides that hybridize to 
the herein above-described sequences. In this regard, the present 
invention especially relates to polynucleotides which hybridize under 
stringent conditions to the herein above-described polynucleotides. As 
herein used, the term "stringent conditions" means hybridization will 
occur only if there is at least 95% and preferably at least 97% identity 
between the sequences. 
As discussed additionally herein regarding polynucleotide assays of the 
invention, for instance, polynucleotides of the invention as discussed 
above, may be used as a hybridization probe for cDNA and genomic DNA to 
isolate full-length cDNAs and genomic clones encoding CGRP-RCF and to 
isolate cDNA and genomic clones of other genes that have a high sequence 
similarity to the human CGRP-RCF gene. Such probes generally will comprise 
at least 15 bases. Preferably, such probes will have at least 30 bases and 
may have at least 50 bases. Particularly preferred probes will have at 
least 30 bases and will have 50 bases or less. 
For example, the coding region of the CGRP-RCF gene may be isolated by 
screening using the known DNA sequence to synthesize an oligonucleotide 
probe. A labeled oligonucleotide having a sequence complementary to that 
of a gene of the present invention is then used to screen a library of 
human cDNA, genomic DNA or mRNA to determine which members of the library 
the probe hybridizes to. 
The polynucleotides and polypeptides of the present invention may be 
employed as research reagents and materials for discovery of treatments 
and diagnostics to human disease, as further discussed herein relating to 
polynucleotide assays, inter alia. 
The polynucleotides may encode a polypeptide which is the mature protein 
plus additional amino or carboxyl-terminal amino acids, or amino acids 
interior to the mature polypeptide (when the mature form has more than one 
polypeptide chain, for instance). Such sequences may play a role in 
processing of a protein from precursor to a mature form, may facilitate 
protein trafficking, may prolong or shorten protein half-life or may 
facilitate manipulation of a protein for assay or production, among other 
things. As generally is the case in situ, the additional amino acids may 
be processed away from the mature protein by cellular enzymes. 
A precursor protein, having the mature form of the polypeptide fused to one 
or more prosequences may be an inactive form of the polypeptide. When 
prosequences are removed such inactive precursors generally are activated. 
Some or all of the prosequences may be removed before activation. 
Generally, such precursors are called proproteins. 
In sum, a polynucleotide of the present invention may encode a mature 
protein, a mature protein plus a leader sequence (which may be referred to 
as a preprotein), a precursor of a mature protein having one or more 
prosequences which are not the leader sequences of a preprotein, or a 
preproprotein, which is a precursor to a proprotein, having a leader 
sequence and one or more prosequences, which generally are removed during 
processing steps that produce active and mature forms of the polypeptide. 
Deposited materials 
A deposit containing a human CGRP-RCF cDNA has been deposited with the 
American Type Culture Collection, as noted above. Also as noted above, the 
human cDNA deposit is referred to herein as "the deposited clone" or as 
"the cDNA of the deposited clone." 
The deposited clone is deposited with the American Type Culture Collection, 
12301 Park Lawn Drive, Rockville, Md. 20852, U.S.A., on Jul. 17, 1996, and 
assigned ATCC Deposit No. 98105 (Escherichia coli pHOUDC44/SolR). 
The deposited material is a pBluescript SK (-) plasmid (Stratagene, La 
Jolla, Calif.)* that contains the full length CGRP-RCF cDNA, referred to 
as pHOUDC44 upon deposit. 
The deposit has been made under the terms of the Budapest Treaty on the 
international recognition of the deposit of micro-organisms for purposes 
of patent procedure. The strain will be irrevocably and without 
restriction or condition released to the public upon the issuance of a 
patent. The deposit is provided merely as convenience to those of skill in 
the art and is not an admission that a deposit is required for enablement, 
such as that required under 35 U.S.C. .sctn.112. 
The sequence of the polynucleotides contained in the deposited material, as 
well as the amino acid sequence of the polypeptide encoded thereby, are 
controlling in the event of any conflict with any description of sequences 
herein. 
A license may be required to make, use or sell the deposited materials, and 
no such license is hereby granted. 
Polypeptides 
The present invention further relates to a human CGRP-RCF polypeptide which 
has the deduced amino acid sequence of FIG. 1. SEQ ID NO:2. 
The invention also relates to fragments, analogs and derivatives of these 
polypeptides. The terms "fragment," "derivative" and "analog" when 
referring to the polypeptide of FIG. 1, means a polypeptide which retains 
essentially the same biological function or activity as such polypeptide, 
i.e. functions as a CGRP-RCF, or retains the ability to bind the CGRP 
receptor ligand or the CGRP receptor even though the polypeptide does not 
function as a CGRP-RCF, for example, a soluble form of CGRP-RCF. Thus, an 
analog includes a proprotein which can be activated by cleavage of the 
proprotein portion to produce an active mature polypeptide. 
The polypeptide of the present invention may be a recombinant polypeptide, 
a natural polypeptide or a synthetic polypeptide. In certain preferred 
embodiments it is a recombinant polypeptide. 
The fragment, derivative or analog of the polypeptide of FIG. 1 may be (i) 
one in which one or more of the amino acid residues are substituted with a 
conserved or non-conserved amino acid residue (preferably a conserved 
amino acid residue) and such substituted amino acid residue may or may not 
be one encoded by the genetic code, or (ii) one in which one or more of 
the amino acid residues includes a substituent group, or (iii) one in 
which the mature polypeptide is fused with another compound, such as a 
compound to increase the half-life of the polypeptide (for example, 
polyethylene glycol), or (iv) one in which the additional amino acids are 
fused to the mature polypeptide, such as a leader or secretory sequence or 
a sequence which is employed for purification of the mature polypeptide or 
a proprotein sequence. Such fragments, derivatives and analogs are deemed 
to be within the scope of those skilled in the art from the teachings 
herein. 
Among the particularly preferred embodiments of the invention in this 
regard are polypeptides having the amino acid sequence of CGRP-RCF set out 
in FIG. 1, variants, analogs, derivatives and fragments thereof, and 
variants, analogs and derivatives of the fragments. Alternatively, 
particularly preferred embodiments of the invention in this regard are 
polypeptides having the amino acid sequence of the CGRP-RCF, variants, 
analogs, derivatives and fragments thereof, and variants, analogs and 
derivatives of the fragments. 
Among preferred variants are those that vary from a reference by 
conservative amino acid substitutions. Such substitutions are those that 
substitute a given amino acid in a polypeptide by another amino acid of 
like characteristics. Typically seen as conservative substitutions are the 
replacements, one for another, among the aliphatic amino acids Ala, Val, 
Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of 
the acidic residues Asp and Glu, substitution between the amide residues 
Asn and Gln, exchange of the basic residues Lys and Arg and replacements 
among the aromatic residues Phe, Tyr. 
Further particularly preferred in this regard are variants, analogs, 
derivatives and fragments, and variants, analogs and derivatives of the 
fragments, having the amino acid sequence of the CGRP-RCF polypeptide of 
FIG. 1, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino 
acid residues are substituted, deleted or added, in any combination. 
Especially preferred among these are silent substitutions, additions and 
deletions, which do not alter the properties and activities of the 
CGRP-RCF. Also especially preferred in this regard are conservative 
substitutions. Most highly preferred are polypeptides having the amino 
acid sequence of FIG. 1 without substitutions. 
The polypeptides and polynucleotides of the present invention are 
preferably provided in an isolated form, and preferably are purified to 
homogeneity. 
The polypeptides of the present invention include the polypeptide of SEQ ID 
NO:2 (in particular the mature polypeptide) as well as polypeptides which 
have at least 80% identity to the polypeptide of SEQ ID NO:2 and more 
preferably at least 90% similarity (more preferably at least 90% identity) 
to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% 
similarity (still more preferably at least 95% identity) to the 
polypeptide of SEQ ID NO:2 and also include portions of such polypeptides 
with such portion of the polypeptide generally containing at least 30 
amino acids and more preferably at least 50 amino acids. 
As known in the art "similarity" between two polypeptides is determined by 
comparing the amino acid sequence and its conserved amino acid substitutes 
of one polypeptide to the sequence of a second polypeptide. Moreover, also 
known in the an is "identity" which means the degree of sequence 
relatedness between two polypeptide or two polynucleotides sequences as 
determined by the identity of the match between two strings of such 
sequences. Both identity and similarity can be readily calculated 
(Computational Molecular Biology, Lesk, A. M., ed., Oxford University 
Press, New York, 1988; Biocomputing: Informatics and Genome Projects, 
Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of 
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana 
Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von 
Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, 
M. and Devereux, J., eds., M. Stockton Press, New York, 1991). While there 
exist a number of methods to measure identity and similarity between two 
polynucleotide or polypeptide sequences, the terms "identity" and 
"similarity" are well known to skilled artisans (Sequence Analysis in 
Molecular Biology, yon Heinje, G., Academic Press, 1987; Sequence Analysis 
Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 
1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 
(1988). Methods commonly employed to determine identity or similarity 
between two sequences include, but are not limited to disclosed in Guide 
to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, 
and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). 
Preferred methods to determine identity are designed to give the largest 
match between the two sequences tested. Methods to determine identity and 
similarity are codified in computer programs. Preferred computer program 
methods to determine identity and similarity between two sequences 
include, but are not limited to, GCG program package (Devereux, J., et 
al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA 
(Atschul, S. F. et al., J. Molec. Biol. 215:403 (1990)). 
Fragments or portions of the polypeptides of the present invention may be 
employed for producing the corresponding full-length polypeptide by 
peptide synthesis; therefore, the fragments may be employed as 
intermediates for producing the full-length polypeptides. Fragments or 
portions of the polynucleotides of the present invention may be used to 
synthesize full-length polynucleotides of the present invention. 
Fragments 
Also among preferred embodiments of this aspect of the present invention 
are polypeptides comprising fragments of CGRP-RCF, most particularly 
fragments of the CGRP-RCF having the amino acid set out in FIG. 1, and 
fragments of variants and derivatives of the CGRP-RCF of FIG. 1. 
In this regard a fragment is a polypeptide having an amino acid sequence 
that entirely is the same as part but not all of the amino acid sequence 
of the aforementioned CGRP-RCF polypeptides and variants or derivatives 
thereof. 
Such fragments may be "free-standing," i.e., not part of or fused to other 
amino acids or polypeptides, or they may be comprised within a larger 
polypeptide of which they form a part or region. When comprised within a 
larger polypeptide, the presently discussed fragments most preferably form 
a single continuous region. However, several fragments may be comprised 
within a single larger polypeptide. For instance, certain preferred 
embodiments relate to a fragment of a CGRP-RCF polypeptide of the present 
comprised within a precursor polypeptide designed for expression in a host 
and having heterologous pre and pro-polypeptide regions fused to the amino 
terminus of the CGRP-RCF fragment and an additional region fused to the 
carboxyl terminus of the fragment. Therefore, fragments in one aspect of 
the meaning intended herein, refers to the portion or portions of a fusion 
polypeptide or fusion protein derived from CGRP-RCF. 
As representative examples of polypeptide fragments of the invention, there 
may be mentioned those which have from about 5-15, 10-20, 15-40, 30-55, 
41-75, 41-80, 41-90, 50-100, 75-100, 90-115, 100-125, and 110-113 amino 
acids long. 
In this context about includes the particularly recited range and ranges 
larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid at either 
extreme or at both extremes. For instance, about 40-90 amino acids in this 
context means a polypeptide fragment of 40 plus or minus several, a few, 
5, 4, 3, 2 or 1 amino acids to 90 plus or minus several a few, 5, 4, 3, 2 
or 1 amino acid residues, i.e., ranges as broad as 40 minus several amino 
acids to 90 plus several amino acids to as narrow as 40 plus several amino 
acids to 90 minus several amino acids. 
Highly preferred in this regard are the recited ranges plus or minus as 
many as 5 amino acids at either or at both extremes. Particularly highly 
preferred are the recited ranges plus or minus as many as 3 amino acids at 
either or at both the recited extremes. Especially particularly highly 
preferred are ranges plus or minus 1 amino acid at either or at both 
extremes or the recited ranges with no additions or deletions. Most highly 
preferred of all in this regard are fragments from about 5-15, 10-20, 
15-40, 30-55, 41-75, 41-80, 41-90, 50-100, 75-100, 90-115, 100-125, and 
110-113 amino acids long. 
Among especially preferred fragments of the invention are truncation 
mutants of CGRP-RCF. Truncation mutants include CGRP-RCF polypeptides 
having the amino acid sequence of FIG. 1, or of variants or derivatives 
thereof, except for deletion of a continuous series of residues (that is, 
a continuous region, part or portion) that includes the amino terminus, or 
a continuous series of residues that includes the carboxyl terminus or, as 
in double truncation mutants, deletion of two continuous series of 
residues, one including the amino terminus and one including the carboxyl 
terminus. Fragments having the size ranges set out about also are 
preferred embodiments of truncation fragments, which are especially 
preferred among fragments generally. 
Also preferred in this aspect of the invention are fragments characterized 
by structural or functional attributes of CGRP-RCF. Preferred embodiments 
of the invention in this regard include fragments that comprise 
alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet 
and beta-sheet-forming regions ("beta-regions"), turn and turn-forming 
regions ("turn-regions"), coil and coil-forming regions ("coil-regions"), 
hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta 
amphipathic regions, flexible regions, surface-forming regions and high 
antigenic index regions of CGRP-RCF. 
Among highly preferred fragments in this regard are those that comprise 
regions of CGRP-RCF that combine several structural features, such as 
several of the features set out above. In this regard, the regions defined 
by the residues about 10 to about 20, about 40 to about 50, about 70 to 
about 90 and about 100 to about 113 of FIG. 1, which all are characterized 
by amino acid compositions highly characteristic of turn-regions, 
hydrophilic regions, flexible-regions, surface-forming regions, and high 
antigenic index-regions, are especially highly preferred regions. Such 
regions may be comprised within a larger polypeptide or may be by 
themselves a preferred fragment of the present invention, as discussed 
above. It will be appreciated that the term "about" as used in this 
paragraph has the meaning set out above regarding fragments in general. 
Further preferred regions are those that mediate activities of CGRP-RCF. 
Most highly preferred in this regard are fragments that have a chemical, 
biological or other activity of CGRP-RCF, including those with a similar 
activity or an improved activity, or with a decreased undesirable 
activity. Highly preferred in this regard are fragments that contain 
regions that are homologs in sequence, or in position, or in both sequence 
and to active regions of related polypeptides, such as the related 
polypeptides of guinea pig CGRP-RCP. Among particularly preferred 
fragments in these regards are truncation mutants, as discussed above. 
It will be appreciated that the invention also relates to, among others, 
polynucleotides encoding the aforementioned fragments, polynucleotides 
that hybridize to polynucleotides encoding the fragments, particularly 
those that hybridize under stringent conditions, and polynucleotides, such 
as PCR primers, for amplifying polynucleotides that encode the fragments. 
In these regards, preferred polynucleotides are those that correspondent 
to the preferred fragments, as discussed above. 
Vectors, host cells, expression 
The present invention also relates to vectors which include polynucleotides 
of the present invention, host cells which are genetically engineered with 
vectors of the invention and the production of polypeptides of the 
invention by recombinant techniques. 
Host cells can be genetically engineered to incorporate polynucleotides and 
express polypeptides of the present invention. For instance, 
polynucleotides may be introduced into host cells using well known 
techniques of infection, transduction, transfection, transvection and 
transformation. The polynucleotides may be introduced alone or with other 
polynucleotides. Such other polynucleotides may be introduced 
independently, co-introduced or introduced joined to the polynucleotides 
of the invention. 
Thus, for instance, polynucleotides of the invention may be transfected 
into host cells with another, separate, polynucleotide encoding a 
selectable marker, using standard techniques for co-transfection and 
selection in, for instance, mammalian cells. In this case the 
polynucleotides generally will be stably incorporated into the host cell 
genome. 
Alternatively, the polynucleotides may be joined to a vector containing a 
selectable marker for propagation in a host. The vector construct may be 
introduced into host cells by the aforementioned techniques. Generally, a 
plasmid vector is introduced as DNA in a precipitate, such as a calcium 
phosphate precipitate, or in a complex with a charged lipid. 
Electroporation also may be used to introduce polynucleotides into a host. 
If the vector is a virus, it may be packaged in vitro or introduced into a 
packaging cell and the packaged virus may be transduced into cells. A wide 
variety of techniques suitable for making polynucleotides and for 
introducing polynucleotides into cells in accordance with this aspect of 
the invention are well known and routine to those of skill in the art. 
Such techniques are reviewed at length in Sambrook et al. cited above, 
which is illustrative of the many laboratory manuals that detail these 
techniques. 
In accordance with this aspect of the invention the vector may be, for 
example, a plasmid vector, a single or double-stranded phage vector, a 
single or double-stranded RNA or DNA viral vector. Such vectors may be 
introduced into cells as polynucleotides, preferably DNA, by well known 
techniques for introducing DNA and RNA into cells. The vectors, in the 
case of phage and viral vectors also may be and preferably are introduced 
into cells as packaged or encapsidated virus by well known techniques for 
infection and transduction. Viral vectors may be replication competent or 
replication defective. In the latter case viral propagation generally will 
occur only in complementing host cells. 
Preferred among vectors, in certain respects, are those for expression of 
polynucleotides and polypeptides of the present invention. Generally, such 
vectors comprise cis-acting control regions effective for expression in a 
host operatively linked to the polynucleotide to be expressed. Appropriate 
trans-acting factors either are supplied by the host, supplied by a 
complementing vector or supplied by the vector itself upon introduction 
into the host. 
In certain preferred embodiments in this regard, the vectors provide for 
specific expression. Such specific expression may be inducible expression 
or expression only in certain types of cells or both inducible and 
cell-specific. Particularly preferred among inducible vectors are vectors 
that can be induced for expression by environmental factors that are easy 
to manipulate, such as temperature and nutrient additives. A variety of 
vectors suitable to this aspect of the invention, including constitutive 
and inducible expression vectors for use in prokaryotic and eukaryotic 
hosts, are well known and employed routinely by those of skill in the art. 
The engineered host cells can be cultured in conventional nutrient media, 
which may be modified as appropriate for, inter alia, activating 
promoters, selecting transformants or amplifying genes. Culture 
conditions, such as temperature, pH and the like, previously used with the 
host cell selected for expression generally will be suitable for 
expression of polypeptides of the present invention as will be apparent to 
those of skill in the art. 
A great variety of expression vectors can be used to express a polypeptide 
of the invention. Such vectors include chromosomal, episomal and 
virus-derived vectors e.g., vectors derived from bacterial plasmids, from 
bacteriophage, from yeast episomes, from yeast chromosomal elements, from 
viruses such as baculoviruses, papova viruses, such as SV40, vaccinia 
viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and 
retroviruses, and vectors derived from combinations thereof, such as those 
derived from plasmid and bacteriophage genetic elements, such as cosmids 
and phagemids, all may be used for expression in accordance with this 
aspect of the present invention. Generally, any vector suitable to 
maintain, propagate or express polynucleotides to express a polypeptide in 
a host may be used for expression in this regard. 
The appropriate DNA sequence may be inserted into the vector by any of a 
variety of well-known and routine techniques. In general, a DNA sequence 
for expression is joined to an expression vector by cleaving the DNA 
sequence and the expression vector with one or more restriction 
endonucleases and then joining the restriction fragments together using T4 
DNA ligase. Procedures for restriction and ligation that can be used to 
this end are well known and routine to those of skill. Suitable procedures 
in this regard, and for constructing expression vectors using alternative 
techniques, which also are well known and routine to those skilled in the 
art, are set forth in great detail in Sambrook et al. cited elsewhere 
herein. 
The DNA sequence in the expression vector is operatively linked to 
appropriate expression control sequence(s), including, for instance, a 
promoter to direct mRNA transcription. Representatives of such promoters 
include the phage lambda PL promoter, the E. coli lac, trp and tac 
promoters, the SV40 early and late promoters and promoters of retroviral 
LTRs, to name just a few of the well-known promoters. It will be 
understood that numerous promoters not mentioned are suitable for use in 
this aspect of the invention are well known and readily may be employed by 
those of skill in the manner illustrated by the discussion and the 
examples herein. 
In general, expression constructs will contain sites for transcription 
initiation and termination, and, in the transcribed region, a ribosome 
binding site for translation. The coding portion of the mature transcripts 
expressed by the constructs will include a translation initiating AUG at 
the beginning and a termination codon appropriately positioned at the end 
of the polypeptide to be translated. 
In addition, the constructs may contain control regions that regulate as 
well as engender expression. Generally, in accordance with many commonly 
practiced procedures, such regions will operate by controlling 
transcription, such as repressor binding sites and enhancers, among 
others. 
Vectors for propagation and expression generally will include selectable 
markers. Such markers also may be suitable for amplification or the 
vectors may contain additional markers for this purpose. In this regard, 
the expression vectors preferably contain one or more selectable marker 
genes to provide a phenotypic trait for selection of transformed host 
cells. Preferred markers include dihydrofolate reductase or neomycin 
resistance for eukaryotic cell culture, and tetracycline or ampicillin 
resistance genes for culturing E. coli and other bacteria. 
The vector containing the appropriate DNA sequence as described elsewhere 
herein, as well as an appropriate promoter, and other appropriate control 
sequences, may be introduced into an appropriate host using a variety of 
well known techniques suitable to expression therein of a desired 
polypeptide. Representative examples of appropriate hosts include 
bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium 
cells; fungal cells, such as yeast cells; insect cells such as Drosophila 
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes 
melanoma cells; and plant cells. Hosts for of a great variety of 
expression constructs are well known, and those of skill will be enabled 
by the present disclosure readily to select a host for expressing a 
polypeptides in accordance with this aspect of the present invention. 
More particularly, the present invention also includes recombinant 
constructs, such as expression constructs, comprising one or more of the 
sequences described above. The constructs comprise a vector, such as a 
plasmid or viral vector, into which such a sequence of the invention has 
been inserted. The sequence may be inserted in a forward or reverse 
orientation. In certain preferred embodiments in this regard, the 
construct further comprises regulatory sequences, including, for example, 
a promoter, operably linked to the sequence. Large numbers of suitable 
vectors and promoters are known to those of skill in the art, and there 
are many commercially available vectors suitable for use in the present 
invention. 
The following vectors, which are commercially available, are provided by 
way of example. Among vectors preferred for use in bacteria are pQE70, 
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, 
Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from 
Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from 
Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, 
pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL 
available from Pharmacia. These vectors are listed solely by way of 
illustration of the many commercially available and well known vectors 
that are available to those of skill in the art for use in accordance with 
this aspect of the present invention. It will be appreciated that any 
other plasmid or vector suitable for, for example, introduction, 
maintenance, propagation or expression of a polynucleotide or polypeptide 
of the invention in a host may be used in this aspect of the invention. 
Promoter regions can be selected from any desired gene using vectors that 
contain a reporter transcription unit lacking a promoter region, such as a 
chloramphenicol acetyl transferase ("CAT") transcription unit, downstream 
of restriction site or sites for introducing a candidate promoter 
fragment; i.e., a fragment that may contain a promoter. As is well known, 
introduction into the vector of a promoter-containing fragment at the 
restriction site upstream of the cat gene engenders production of CAT 
activity, which can be detected by standard CAT assays. Vectors suitable 
to this end are well known and readily available. Two such vectors are 
pKK232-8 and pCM7. Thus, promoters for expression of polynucleotides of 
the present invention include not only well known and readily available 
promoters, but also promoters that readily may be obtained by the 
foregoing technique, using a reporter gene. 
Among known bacterial promoters suitable for expression of polynucleotides 
and polypeptides in accordance with the present invention are the E. coli 
lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the 
lambda PR, PL promoters and the trp promoter. 
Among known eukaryotic promoters suitable in this regard are the CMV 
immediate early promoter, the HSV thymidine kinase promoter, the early and 
late SV40 promoters, the promoters of retroviral LTRs, such as those of 
the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the 
mouse metallothionein-I promoter. 
Selection of appropriate vectors and promoters for expression in a host 
cell is a well known procedure and the requisite techniques for expression 
vector construction, introduction of the vector into the host and 
expression in the host are routine skills in the art. 
The present invention also relates to host cells containing the 
above-described constructs discussed above. The host cell can be a higher 
eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, 
such as a yeast cell, or the host cell can be a prokaryotic cell, such as 
a bacterial cell. 
Introduction of the construct into the host cell can be effected by calcium 
phosphate transfection, DEAE-dextran mediated transfection, cationic 
lipid-mediated transfection, electroporation, transduction, infection or 
other methods. Such methods are described in many standard laboratory 
manuals, such as Davis et al. BASIC METHODS IN MOLECULAR BIOLOGY, (1986). 
Constructs in host cells can be used in a conventional manner to produce 
the gene product encoded by the recombinant sequence. Alternatively, the 
polypeptides of the invention can be synthetically produced by 
conventional peptide synthesizers. 
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or 
other cells under the control of appropriate promoters. Cell-free 
translation systems can also be employed to produce such proteins using 
RNAs derived from the DNA constructs of the present invention. Appropriate 
cloning and expression vectors for use with prokaryotic and eukaryotic 
hosts are described by Sambrook et al., MOLECULAR CLONING: A LABORATORY 
MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 
N.Y. (1989). 
Generally, recombinant expression vectors will include origins of 
replication, a promoter derived from a highly-expressed gene to direct 
transcription of a downstream structural sequence, and a selectable marker 
to permit isolation of vector containing cells after exposure to the 
vector. Among suitable promoters are those derived from the genes that 
encode glycolytic enzymes such as 3-phosphoglycerate kinase ("PGK"), 
a-factor, acid phosphatase, and heat shock proteins, among others. 
Selectable markers include the ampicillin resistance gene of E. coli and 
the trp 1 gene of S. cerevisiae. 
Transcription of the DNA encoding the polypeptides of the present invention 
by higher eukaryotes may be increased by inserting an enhancer sequence 
into the vector. Enhancers are cis-acting elements of DNA, usually about 
from 10 to 300 bp that act to increase transcriptional activity of a 
promoter in a given host cell-type. Examples of enhancers include the SV40 
enhancer, which is located on the late side of the replication origin at 
bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma 
enhancer on the late side of the replication origin, and adenovirus 
enhancers. 
Polynucleotides of the invention, encoding the heterologous structural 
sequence of a polypeptide of the invention generally will be inserted into 
the vector using standard techniques so that it is operably linked to the 
promoter for expression. The polynucleotide will be positioned so that the 
transcription start site is located appropriately 5' to a ribosome binding 
site. The ribosome binding site will be 5' to the AUG that initiates 
translation of the polypeptide to be expressed. Generally, there will be 
no other open reading frames that begin with an initiation codon, usually 
AUG, and lie between the ribosome binding site and the initiating AUG. 
Also, generally, there will be a translation stop codon at the end of the 
polypeptide and there will be a polyadenylation signal and a transcription 
termination signal appropriately disposed at the 3' end of the transcribed 
region. 
For secretion of the translated protein into the lumen of the endoplasmic 
reticulum, into the periplasmic space or into the extracellular 
environment, appropriate secretion signals may be incorporated into the 
expressed polypeptide. The signals may be endogenous to the polypeptide or 
they may be heterologous signals. 
The polypeptide may be expressed in a modified form, such as a fusion 
protein, and may include not only secretion signals but also additional 
heterologous functional regions. Thus, for instance, a region of 
additional amino acids, particularly charged amino acids, may be added to 
the N-terminus of the polypeptide to improve stability and persistence in 
the host cell, during purification or during subsequent handling and 
storage. Also, region also may be added to the polypeptide to facilitate 
purification. Such regions may be removed prior to final preparation of 
the polypeptide. The addition of peptide moieties to polypeptides to 
engender secretion or excretion, to improve stability and to facilitate 
purification, among others, are familiar and routine techniques in the 
art. 
Suitable prokaryotic hosts for propagation, maintenance or expression of 
polynucleotides and polypeptides in accordance with the invention include 
Escherichia coli, Bacillus subtilis and Salmonella typhimurium. Various 
species of Pseudomonas, Streptomyces, and Staphylococcus are suitable 
hosts in this regard. Moreover, many other hosts also known to those of 
skill may be employed in this regard. 
As a representative but non-limiting example, useful expression vectors for 
bacterial use can comprise a selectable marker and bacterial origin of 
replication derived from commercially available plasmids comprising 
genetic elements of the well known cloning vector pBR322 (ATCC 37017). 
Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine 
Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., 
U.S.A.). These pBR322 "backbone" sections are combined with an appropriate 
promoter and the structural sequence to be expressed. 
Following transformation of a suitable host strain and growth of the host 
strain to an appropriate cell density, where the selected promoter is 
inducible it is induced by appropriate means (e.g., temperature shift or 
exposure to chemical inducer) and cells are cultured for an additional 
period. 
Cells typically then are harvested by centrifugation, disrupted by physical 
or chemical means, and the resulting crude extract retained for further 
purification. 
Microbial cells employed in expression of proteins can be disrupted by any 
convenient method, including freeze-thaw cycling, sonication, mechanical 
disruption, or use of cell lysing agents, such methods are well know to 
those skilled in the art. 
Various mammalian cell culture systems can be employed for expression, as 
well. Examples of mammalian expression systems include the COS-7 lines of 
monkey kidney fibroblast, described in Gluzman et al., Cell 23: 175 
(1981). Other cell lines capable of expressing a compatible vector include 
for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell 
lines. 
Mammalian expression vectors will comprise an origin of replication, a 
suitable promoter and enhancer, and also any necessary ribosome binding 
sites, polyadenylation sites, splice donor and acceptor sites, 
transcriptional termination sequences, and 5' flanking non-transcribed 
sequences that are necessary for expression. In certain preferred 
embodiments in this regard DNA sequences derived from the SV40 splice 
sites, and the SV40 polyadenylation sites are used for required 
non-transcribed genetic elements of these types. 
The CGRP-RCF polypeptide can be recovered and purified from recombinant 
cell cultures by well-known methods including ammonium sulfate or ethanol 
precipitation, acid extraction, anion or cation exchange chromatography, 
phosphocellulose chromatography, hydrophobic interaction chromatography, 
affinity chromatography, hydroxylapatite chromatography and lectin 
chromatography. Most preferably, high performance liquid chromatography 
("HPLC") is employed for purification. Well known techniques for refolding 
protein may be employed to regenerate active conformation when the 
polypeptide is denatured during isolation and or purification. 
Polypeptides of the present invention include naturally purified products, 
products of chemical synthetic procedures, and products produced by 
recombinant techniques from a prokaryotic or eukaryotic host, including, 
for example, bacterial, yeast, higher plant, insect and mammalian cells. 
Depending upon the host employed in a recombinant production procedure, 
the polypeptides of the present invention may be glycosylated or may be 
non-glycosylated. In addition, polypeptides of the invention may also 
include an initial modified methionine residue, in some cases as a result 
of host-mediated processes. 
CGRP-RCF polynucleotides and polypeptides may be used in accordance with 
the present invention for a variety of applications, particularly those 
that make use of the chemical and biological properties of CGRP-RCF. 
Additional applications relate to diagnosis and to treatment of disorders 
of cells, tissues and organisms. These aspects of the invention are 
illustrated further by the following discussion. 
Polynucleotide assays 
This invention is also related to the use of the CGRP-RCF polynucleotides 
to detect complementary polynucleotides such as, for example, as a 
diagnostic reagent. Detection of a mutated form of CGRP-RCF associated 
with a dysfunction will provide a diagnostic tool that can add or define a 
diagnosis of a disease or susceptibility to a disease which results from 
under-expression over-expression or altered expression of CGRP-RCF. 
Individuals carrying mutations in the human CGRP-RCF gene may be detected 
at the DNA level by a variety of techniques. Nucleic acids for diagnosis 
may be obtained from a patient's cells, such as from blood, urine, saliva, 
tissue biopsy and autopsy material. The genomic DNA may be used directly 
for detection or may be amplified enzymatically by using PCR prior to 
analysis. PCR (Saiki et al., Nature, 324: 163-166 (1986)). RNA or cDNA may 
also be used in the same ways. As an example, PCR primers complementary to 
the nucleic acid encoding CGRP-RCF can be used to identify and analyze 
CGRP-RCF expression and mutations. For example, deletions and insertions 
can be detected by a change in size of the amplified product in comparison 
to the normal genotype. Point mutations can be identified by hybridizing 
amplified DNA to radiolabeled CGRP-RCF RNA or alternatively, radiolabeled 
CGRP-RCF antisense DNA sequences. Perfectly matched sequences can be 
distinguished from mismatched duplexes by RNase A digestion or by 
differences in melting temperatures. 
Sequence differences between a reference gene and genes having mutations 
also may be revealed by direct DNA sequencing. In addition, cloned DNA 
segments may be employed as probes to detect specific DNA segments. The 
sensitivity of such methods can be greatly enhanced by appropriate use of 
PCR or another amplification method. For example, a sequencing primer is 
used with double-stranded PCR product or a single-stranded template 
molecule generated by a modified PCR. The sequence determination is 
performed by conventional procedures with radiolabeled nucleotide or by 
automatic sequencing procedures with fluorescent-tags. 
Genetic testing based on DNA sequence differences may be achieved by 
detection of alteration in electrophoretic mobility of DNA fragments in 
gels, with or without denaturing agents. Small sequence deletions and 
insertions can be visualized by high resolution gel electrophoresis. DNA 
fragments of different sequences may be distinguished on denaturing 
formamide gradient gels in which the mobilities of different DNA fragments 
are retarded in the gel at different positions according to their specific 
melting or partial melting temperatures (see, e.g., Myers et al., Science 
230: 1242 (1985)). 
Sequence changes at specific locations also may be revealed by nuclease 
protection assays, such as RNase and S1 protection or the chemical 
cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 
85:4397-4401 (1985)). 
Thus, the detection of a specific DNA sequence may be achieved by methods 
such as hybridization, RNase protection, chemical cleavage, direct DNA 
sequencing or the use of restriction enzymes, (e.g., restriction fragment 
length polymorphisms ("RFLP") and Southern blotting of genomic DNA. 
In accordance with a further aspect of the invention, there is provided a 
process for determining or diagnosing diabetes, migrane, pain and 
inflammation, Parkinson's disease, acute heart failure, hypotension, 
urinary retention, osteoporosis, hypertension, angina pectoris, myocardial 
infarction, ulcers, asthma, allergies, psychosis, depression, vomiting, 
benign prostatic hypertrophy, Paget's disease, obesity, cancer, gigantism 
and the like or a susceptibility to the foregoing diseases. Thus, a 
mutation in CGRP-RCF indicates a susceptibility to treat diabetes, 
migrane, pain and intimation, Parkinson's disease, acute heart failure, 
hypotension, urinary retention, osteoporosis, hypertension, angina 
pectoris, myocardial infarction, ulcers, asthma, allergies, psychosis, 
depression, vomiting, benign prostatic hypertrophy, Paget's disease, 
obesity, cancer, gigantism and the like, and the nucleic acid sequences 
described above may be employed in an assay for ascertaining such 
susceptibility. Thus, for example, the assay may be employed to determine 
a mutation in a human CGRP-RCF protein as herein described, such as a 
deletion, truncation, insertion, frame shift, etc., with such mutation 
being indicative of a susceptibility to the foregoing diseases. 
A mutation may be ascertained for example, by a DNA sequencing assay. 
Tissue samples, including but not limited to blood samples are obtained 
from a human patient. The samples are processed by methods known in the 
art to capture the RNA. First strand cDNA is synthesized from the RNA 
samples by adding an oligonucleotide primer consisting of polythymidine 
residues which hybridize to the polyadenosine stretch present on the 
mRNA's. Reverse transcriptase and deoxynucleotides are added to allow 
synthesis of the first strand cDNA. Primer sequences are synthesized based 
on the DNA sequence of the protein of the invention. The primer sequence 
is generally comprised of at least 15 consecutive bases, and may contain 
at least 30 or even 50 consecutive bases. 
Thus, the detection of a specific DNA sequence and/or quantitation of the 
level of the sequence may be achieved by methods such as hybridization, 
RNase protection, chemical cleavage, direct DNA sequencing or the use of 
restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms 
(RFLP)) and Southern blotting of genomic DNA. The invention provides a 
process for diagnosing diabetes, migrane, pain and inflammation, 
Parkinson's disease, acute heart failure, hypotension, urinary retention, 
osteoporosis, hypertension, angina pectoris, myocardial infarction, 
ulcers, asthma, allergies, psychosis, depression, vomiting, benign 
prostatic hypertrophy, Paget's disease, obesity, cancer, gigantism and the 
like, comprising determining from a sample derived from a patient an 
abnormally decreased or increased level of expression of polynucleotide 
having the sequence of FIG. 1 (SEQ ID NO: 1). Decreased or increased 
expression of polynucleotide can be measured using any on of the methods 
well known in the art for the quantation of polynucleotides, such as, for 
example, PCR, RT-PCR, RNase protection, Northern blotting and other 
hybridization methods. 
In addition to more conventional gel-electrophoresis and DNA sequencing, 
mutations can also be detected by in situ analysis. 
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase 
chromosomal spread can be used to provide a precise chromosomal location. 
As an example of how this is performed, CGRP-RCF DNA is digested and 
purified with QIAEX II DNA purification kit (QIAGEN, Inc., Chatsworth, 
Calif.) and ligated to Super Cos1 cosmid vector (STRATAGENE, La Jolla, 
Calif.). DNA is purified using Qiagen Plasmid Purification Kit (QIAGEN 
Inc., Chatsworth, Calif.) and 1 mg is labeled by nick translation in the 
presence of Biotin-dATP using BioNick Labeling Kit (GibcoBRL, Life 
Technologies Inc., Gaithersburg, Md.). Biotinilation is detected with 
GENETECT Detection System (CLONTECH Laboratories, Inc. Palo Alto, Calif.). 
In situ Hybridization is performed on slides using ONCOR Light 
Hybridization Kit (ONCOR, Gaithersberg, Md.) to detect single copy 
sequences on metaphase chromosomes. Peripheral blood of normal donors is 
cultured for three days in RPMI 1640 supplemented with 20% FCS, 3% PHA and 
penicillin/streptomycin, synchronized with 10.sup.-7 M methotrexate for 17 
hours and washed twice with unsupplemented RPMI. Cells are incubated with 
10.sup.-3 M thymidine for 7 hours. The cells are arrested in metaphase 
after 20 minutes incubation with colcemid (0.5 .mu.g/ml) followed by 
hypotonic lysis in 75 mM KCl for 15 minutes at 37.degree. C. Cell pellets 
are then spun out and fixed in Carnoy's fixative (3:1 methanol/acetic 
acid). 
Metaphase spreads are prepared by adding a drop of the suspension onto 
slides and aid dried. Hybridization is performed by adding 100 ng of probe 
suspended in 10 ml of hybridization mix (50% formamide, 2.times.SSC, 1% 
dextran sulfate) with blocking human placental DNA 1 .mu.g/ml), Probe 
mixture is denatured for 10 minutes in 70.degree. C. water bath and 
incubated for 1 hour at 37.degree. C., before placing on a prewarmed 
(37.degree. C. ) slide, which is previously denatured in 70% 
formamide/2.times.SSC at 70.degree. C., and dehydrated in ethanol series, 
chilled to 4.degree. C. 
Slides are incubated for 16 hours at 37.degree. C. in a humidified chamber. 
Slides are ished in 50% formamide/2.times.SSC for 10 minutes at 41.degree. 
C. and 2.times.SSC for 7 minutes at 37.degree. C. Hybridization probe is 
detected by incubation of the slides with FITC-Avidin (ONCOR, 
Gaithersberg, Md.), according to the manufacturer protocol. Chromosomes 
are counterstained with propridium iodine suspended in mounting medium. 
Slides are visualized using a Leitz ORTHOPLAN 2-epifluorescence microscope 
and five computer images are taken using Imagenetics Computer and 
Macintosh printer. 
Once a sequence has been mapped to a precise chromosomal location, the 
physical position of the sequence on the chromosome can be correlated with 
genetic map data. Such data are found, for example, in V. McKusick, 
Mendelian Inheritance in Man, which is publicly available on line via 
computer. The relationship between genes and diseases that have been 
mapped to the same chromosomal region are then identified through linkage 
analysis (Co-Inheritance of Physically Adjacent Genes). 
Next, it is necessary to determine the differences in the cDNA or genomic 
sequence between affected and unaffected individuals. If a mutation is 
observed in some or all of the affected individuals but not in any normal 
individuals, then the mutation is likely to be the causative agent of the 
disease. 
Chromosome assays 
The sequences of the present invention are also valuable for chromosome 
identification. The sequence is specifically targeted to and can hybridize 
with a particular location on an individual human chromosome. Moreover, 
there is a current need for identifying particular sites on the 
chromosome. Few chromosome marking reagents based on actual sequence data 
(repeat polymorphisms) are presently available for marking chromosomal 
location. The mapping of DNAs to chromosomes according to the present 
invention is an important first step in correlating those sequences with 
genes associated with disease. 
In certain preferred embodiments in this regard, the cDNA herein disclosed 
is used to clone genomic DNA of a CGRP-RCF gene. This can be accomplished 
using a variety of well known techniques and libraries, which generally 
are available commercially. The genomic DNA the is used for in situ 
chromosome mapping using well known techniques for this purpose. 
Typically, in accordance with routine procedures for chromosome mapping, 
some trial and error may be necessary to identify a genomic probe that 
gives a good in situ hybridization signal. 
In some cases, in addition, sequences can be mapped to chromosomes by 
preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer 
analysis of the 3' untranslated region of the gene is used to rapidly 
select primers that do not span more than one exon in the genomic DNA, 
because primers that span more than one exon in the genomic DNA could 
complicate the amplification process. These primers are then used for PCR 
screening of somatic cell hybrids containing individual human chromosomes. 
Only those hybrids containing the human gene corresponding to the primer 
will yield an amplified fragment. 
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a 
particular DNA to a particular chromosome. Using the present invention 
with the same oligonucleotide primers, sublocalization can be achieved 
with panels of fragments from specific chromosomes or pools of large 
genomic clones in an analogous manner. Other mapping strategies that can 
similarly be used to map to its chromosome include in situ hybridization, 
prescreening with labeled flow-sorted chromosomes and preselection by 
hybridization to construct chromosome specific-cDNA libraries. 
Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphase 
chromosomal spread, as described above, can be used to provide a precise 
chromosomal location in one step. This technique can be used with cDNA as 
short as 50 or 60. For a review of this technique, see Verma et al., HUMAN 
CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York 
(1988). 
With current resolution of physical mapping and genetic mapping techniques, 
a cDNA precisely localized to a chromosomal region associated with the 
disease could be one of between 50 and 500 potential causative genes. 
(This assumes 1 megabase mapping resolution and one gene per 20 kb). 
Polypeptide assays 
The present invention also relates to a diagnostic assays such as 
quantitative and diagnostic assays for detecting levels of CGRP-RCF 
protein in cells and tissues, including determination of normal and 
abnormal levels. Thus, for instance, a diagnostic assay in accordance with 
the invention for detecting over-expression of CGRP-RCF protein compared 
to normal control tissue samples may be used to detect the presence of 
diabetes, migrane, pain and inflammation, Parkinson's disease, acute heart 
failure, hypotension, urinary retention, osteoporosis, hypertension, 
angina pectoris, myocardial infarction, ulcers, asthma, allergies, 
psychosis, depression, vomiting, benign prostatic hypertrophy, Paget's 
disease, obesity, cancer, gigantism and the like, for example. Assay 
techniques that can be used to determine levels of a protein, such as an 
CGRP-RCF protein of the present invention, in a sample derived from a host 
are well-known to those of skill in the art. Such assay methods include 
radioimmunoassays, competitive-binding assays, Western Blot analysis and 
ELISA assays. Among these ELISAs frequently are preferred. An ELISA assay 
initially comprises preparing an antibody specific to CGRP-RCF, preferably 
a monoclonal antibody. In addition a reporter antibody generally is 
prepared which binds to the monoclonal antibody. The reporter antibody is 
attached a detectable reagent such as radioactive, fluorescent or 
enzymatic reagent, in this example horseradish peroxidase enzyme. 
To carry out an ELISA a sample is removed from a host and incubated on a 
solid support, e.g. a polystyrene dish, that binds the proteins in the 
sample. Any free protein binding sites on the dish are then covered by 
incubating with a non-specific protein such as bovine serum albumin. Next, 
the monoclonal antibody is incubated in the dish during which time the 
monoclonal antibodies attach to any CGRP-RCF proteins attached to the 
polystyrene dish. Unbound monoclonal antibody is ished out with buffer. 
The reporter antibody linked to horseradish peroxidase is placed in the 
dish resulting in binding of the reporter antibody to any monoclonal 
antibody bound to CGRP-RCF. Unattached reporter antibody is then ished 
out. Reagents for peroxidase activity, including a colorimetric substrate 
are then added to the dish. Immobilized peroxidase, linked to CGRP-RCF 
through the primary and secondary antibodies, produces a colored reaction 
product. The amount of color developed in a given time period indicates 
the amount of CGRP-RCF protein present in the sample. Quantitative results 
typically are obtained by reference to a standard curve. 
A competition assay may be employed wherein antibodies specific to CGRP-RCF 
attached to a solid support and labeled CGRP-RCF and a sample derived from 
the host are passed over the solid support and the amount of label 
detected attached to the solid support can be correlated to a quantity of 
CGRP-RCF in the sample. 
Antibodies 
The polypeptides, their fragments or other derivatives, or analogs thereof, 
or cells expressing them can be used as an immunogen to produce antibodies 
thereto. These antibodies can be, for example, polyclonal or monoclonal 
antibodies. The present invention also includes chimeric, single chain, 
and humanized antibodies, as well as Fab fragments, or the product of an 
Fab expression library. Various procedures known in the art may be used 
for the production of such antibodies and fragments. 
Antibodies generated against the polypeptides corresponding to a sequence 
of the present invention can be obtained by direct injection of the 
polypeptides into an animal or by administering the polypeptides to an 
animal, preferably a nonhuman. The antibody so obtained will then bind the 
polypeptides itself. In this manner, even a sequence encoding only a 
fragment of the polypeptides can be used to generate antibodies binding 
the whole native polypeptides. Such antibodies can then be used to isolate 
the polypeptide from tissue expressing that polypeptide. 
For preparation of monoclonal antibodies, any technique which provides 
antibodies produced by continuous cell line cultures can be used. Examples 
include the hybridoma technique (Kohler, et al., Nature 256:495-497 
(1975), the trioma technique, the human B-cell hybridoma technique (Kozbor 
et al., Immunology Today 4:72 (1983) and the EBV-hybridoma technique to 
produce human monoclonal antibodies (Cole et al., pg. 77-96 in MONOCLONAL 
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985)). 
Techniques described for the production of single chain antibodies (U.S. 
Pat. No. 4,946,778) can be adapted to produce single chain antibodies to 
immunogenic polypeptide products of this invention. Also, transgenic mice, 
or other organisms such as other mammals, may be used to express humanized 
antibodies to immunogenic polypeptide products of this invention. 
The above-described antibodies may be employed to isolate or to identify 
clones expressing the polypeptide or purify the polypeptide of the present 
invention by attachment of the antibody to a solid support for isolation 
and/or purification by affinity chromatography. 
Thus, among others, antibodies against CGRP-RCF may be employed to inhibit 
diabetes, migrane, pain and inflammation, Parkinson's disease, acute heart 
failure, hypotension, urinary retention, osteoporosis, hypertension, 
angina pectoris, myocardial infarction, ulcers, asthma, allergies, 
psychosis, depression, vomiting, benign prostatic hypertrophy, Paget's 
disease, obesity, cancer, gigantism and the like. 
CGRP-RCF binding molecules and assays 
This invention also provides a method for identification of molecules, such 
as binding molecules, that bind CGRP-RCF/CGRP receptor. Genes encoding 
proteins that bind CGRP-RCF-CGRP receptor, such as binding molecules, can 
be identified by numerous methods known to those of skill in the art, for 
example, ligand panning and FACS sorting. Such methods are described in 
many laboratory manuals such as, for instance, Coligan et al., Current 
Protocols in Immunology 1(2):Chapter 5 (1991). 
For instance, expression cloning may be employed for this purpose. To this 
end polyadenylated RNA is prepared from a cell responsive to CGRP-RCF and 
CGRP receptor, a cDNA library is created from this RNA, the library is 
divided into pools and the pools are transfected individually into cells 
that are not responsive to .alpha.- or .beta.-CGRP. The transfected cells 
then are exposed to labeled .alpha.- or .beta.-CGRP. (.alpha.- or 
.beta.-CGRP can be labeled by a variety of well-known techniques including 
standard methods of radio-iodination or inclusion of a recognition site 
for a site-specific protein kinase.) Following exposure, the cells are 
fixed and binding of .alpha.- or .beta.-CGRP is determined. These 
procedures conveniently are carried out on glass slides. 
Pools are identified of cDNA that produced .alpha.- or .beta.-CGRP binding 
cells. Sub-pools are prepared from these positives, transfected into host 
cells and screened as described above. Using an iterative sub-pooling and 
re-screening process, one or more single clones that encode the putative 
binding molecule, such as a receptor molecule, can be isolated. 
Alternatively a labeled ligand can be photoaffinity linked to a cell 
extract, such as a membrane or a membrane extract, prepared from cells 
that express a molecule that it binds, such as a receptor molecule. 
Cross-linked material is resolved by polyacrylamide gel electrophoresis 
("PAGE") and exposed to X-ray film. The labeled complex containing the 
ligand-receptor can be excised, resolved into peptide fragments, and 
subjected to protein microsequencing. The amino acid sequence obtained 
from microsequencing can be used to design unique or degenerate 
oligonucleotide probes to screen cDNA libraries to identify genes encoding 
the putative binding molecule. 
Polypeptides of the invention also can be used to assess CGRP-RCF binding 
capacity of CGRP-RCF binding molecules in cells or in cell-free 
preparations. 
Agonists and antagonists--assays and molecules 
The CGRP-RCF of the present invention may be employed in a process for 
screening for compounds which activate (agonists) or inhibit activation 
(antagonists) of the CGRP receptor or (function of) CGRP-RCF or even 
CGRP-RCF/CGRP receptor system. 
In general, such screening procedures involve providing appropriate cells 
which express the CGRP-RCF/CGRP receptor on the surface thereof. Such 
cells include cells from mammals, yeast, drosophila or E. Coli. In 
particular, polynucleotides encoding the receptor system of the present 
invention are employed to transfect cells to thereby express the 
CGRP-RCF/CGRP receptor. The expressed polypeptides are then contacted with 
a test compound to observe binding, stimulation or inhibition of a 
functional response. 
One such screening procedure involves the use of melanophores which are 
transfected to express the CGRP-RCF/CGRP receptor. Such a screening 
technique is described in PCT WO 92/01810 published Feb. 6, 1992. 
Thus, for example, such assay may be employed for screening for a compound 
which inhibits activation of the CGRP-RCF/CGRP receptor by contacting the 
melanophore cells which encode the polypeptides with both the CGRP 
receptor ligand and a compound to be screened. Inhibition of the signal 
generated by the ligand indicates that a compound is a potential 
antagonist for the receptor system, i.e., inhibits activation of the 
CGRP-RCF/CGRP receptor. 
The screen may be employed for determining a compound which activates the 
receptor system by contacting such cells with compounds to be screened and 
determining whether such compound generates a signal, i.e., activates the 
CGRP-RCF/CGRP receptor. 
Other screening techniques include the use of cells which express the 
CGRP-RCF-CGRP receptor (for example, transfected CHO cells) in a system 
which measures extracellular pH changes caused by CGRP-RCF/CGRP receptor 
activation, for example, as described in Science, 246:181-296 (October 
1989). For example, compounds may be contacted with a cell which expresses 
the CGRP-RCF/CGRP receptor and a second messenger response, e.g. signal 
transduction or pH changes, may be measured to determine whether the 
potential compound activates or inhibits the CGRP-RCF/CGRP receptor. 
Another such screening technique involves introducing RNAs encoding both 
CGRP-RCF and CGRP receptor into Xenopus oocytes to transiently express the 
CGRP-RCF/CGRP receptor. The oocytes may then be contacted with the CGRP 
receptor ligand and a compound to be screened, followed by detection of 
inhibition or activation of a calcium, proton, etc. signal in the case of 
screening for compounds which are thought to inhibit activation of the 
receptor. 
Another screening technique involves expressing the CGRP-RCF/CGRP receptor 
in which the receptor system is linked to, for example, a phospholipase C 
or D or other proteins. As representative examples of such cells, there 
may be mentioned endothelial cells, smooth muscle cells, embryonic kidney 
cells, etc. The screening may be accomplished as hereinabove described by 
detecting activation of the receptor system or inhibition of activation of 
the receptor system from a second signal, such as for example 
phospholipase or other activated/expressed protein. 
Another method involves screening for compounds which inhibit activation of 
the receptor system of the present invention antagonists by determining 
inhibition of binding of labeled ligand to cells which have the receptor 
system on the surface thereof. Such a method involves transfecting a 
eukaryotic cell with DNAs encoding both CGRP-RCF and CGRP receptor such 
that the cell expresses the receptor system on its surface and contacting 
the cell with a compound in the presence of a labeled form of a known 
ligand. The ligand can be labeled, e.g., by radioactivity. The amount of 
labeled ligand bound to the receptors is measured, e.g., by measuring 
radioactivity of the receptor system. If the compound binds to the 
receptor system as determined by a reduction of labeled ligand which binds 
to the receptor system, the binding of labeled ligand to the receptor 
system is inhibited. 
Another method involves screening for CGRP-RCF/CGRP receptor inhibitors by 
determining inhibition of CGRP-RCF/CGRP receptor-mediated cAMP and/or 
adenylate cyclase accumulation. Such a method involves transfecting a 
eukaryotic cell with the CGRP-RCF/CGRP receptor system to express the 
receptor system on the cell surface. The cell is then exposed to potential 
antagonists in the presence of the receptor system. The amount of cAMP 
accumulation is then measured. If the potential antagonist binds the 
receptor system, and thus inhibits ligand binding, the levels of 
CGRP-RCF/CGRP receptor mediated cAMP, or adenylate cyclase, activity will 
be reduced. 
The present invention also provides a method for determining whether a 
ligand, not known to be capable of binding to a CGRP receptor or 
CGRP-RCF/CGRP receptor, can bind to such receptor or receptor system which 
comprises contacting a mammalian cell which expresses CGRP-RCF/CGRP 
receptor with the CGRP receptor ligand under conditions permitting binding 
of ligands to the CGRP-RCF/CGRP receptor system, detecting the presence of 
a ligand which binds to the receptor and thereby determining whether the 
ligand binds to the CGRP-RCF/CGRP receptor. The systems hereinabove 
described for determining agonists and/or antagonists may also be employed 
for determining ligands which bind to the receptor system. 
Examples of potential CGRP-RCF or CGRP-RCF/CGRP receptor antagonists are 
also an antibody, or in some cases an oligonucleotide, which binds to the 
CGRP-RCF or CGRP-RCF/CGRP receptor such that the activity of the 
polypeptide is prevented. 
Potential antagonists also include proteins which are closely related to 
the ligand of the CGRP-RCF/CGRP receptor, i.e. a fragment of the ligand, 
which have lost biological function and when binding to the receptor 
system, elicit no response. 
A potential antagonist also includes an antisense construct prepared 
through the use of antisense technology. Antisense technology can be used 
to control gene expression through triple-helix formation or antisense DNA 
or RNA, both of which methods are based on binding of a polynucleotide to 
DNA or RNA. For example, the 5' coding portion of the polynucleotide 
sequence, which encodes for the mature polypeptides of the present 
invention, is used to design an antisense RNA oligonucleotide of from 
about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to 
be complementary to a region of the gene involved in transcription (triple 
helix--see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, 
Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), 
thereby preventing transcription and the production of CGRP-RCF. The 
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks 
translation of the mRNA molecule into the CGRP-RCF (antisense--Okano, J. 
Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors 
of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The 
oligonucleotides described above can also be delivered to cells such that 
the antisense RNA or DNA may be expressed in vivo to inhibit production of 
the CGRP-RCF. 
Another potential antagonist is a small organic molecule which binds to the 
CGRP-RCF or CGRP-RCF/CGRP receptor, making it inaccessible to ligands such 
that its normal biological activity is prevented. 
Potential antagonists also include a soluble form of a CGRP-RCF, e.g. a 
fragment, which binds to the CGRP receptor ligand and prevents the ligand 
from interacting with membrane bound CGRP-RCF/CGRP receptor. 
CGRP-RCF are responsible for many biological functions, including many 
pathologies. Accordingly, it is desirous to find compounds and drugs which 
stimulate the CGRP-RCF or CGRP-RCF/CGRP receptor on the one hand and which 
can inhibit the function of a CGRP-RCF or CGRP-RCF/CGRP receptor on the 
other hand. 
In general, agonists for CGRP-RCF or CGRP-RCF/CGRP receptor are employed 
for prophylaxis of or for treating Parkinson's disease, acute heart 
failure, hypotension, urinary retention. 
Antagonists for CGRP-RCF or CGRP-RCF/CGRP receptor may be employed for 
prophylaxis of or for treating hypertension, angina pectoris, myocardial 
infarction, ulcers, asthma, allergies, psychoses, depression, migrane, 
vomiting, and benign prostatic hyertrophy. 
The agonists may also be employed to treat osteoporosis since CGRP inhibits 
osteoclast-mediated bone resorption and stimulates osteogenesis. In this 
same manner hypercalcemia may be treated. Similarly, the agonists may be 
employed to treat Paget's Disease. 
The agonists may also be employed to stimulate angiogenesis and promote 
wound healing via the stimulatory effect of CGRP on endothelial cell 
proliferation. 
The agonists may also be employed to treat obesity since CGRP controls 
feeding behavior by decreasing appetite and intestinal motility. 
The agonists may also be employed to stimulate nerve regeneration since 
CGRP is a trophic agent in the CNS. 
The agonists may also be employed to enhance the immune response through 
increasing vascular permeability. 
Agonists may also be employed to inhibit superoxide production. Superoxide 
production is known in the art to cause cellular damage and lead possibly 
to diseases such as cancer. 
The antagonists may also be employed to inhibit CNS pain transmission, to 
treat chronic inflammation caused by long-lived vasodilation, arthritis, 
maturity onset diabetes, cardiovascular disorders and to treat migraine 
headaches. 
The antagonists may also be employed to prevent carcinoid tumor of the 
lung, since an elevated level of CGRP has been found in the lung during 
this disease. 
This invention additionally provides a method of treating an abnormal 
condition related to an excess of CGRP-RCF activity which comprises 
administering to a subject the inhibitor compounds (antagonists) as 
hereinabove described along with a pharmaceutically acceptable carrier in 
an amount effective to inhibit activation by blocking binding of ligands 
to the CGRP-RCF/CGRP receptor, or by inhibiting a second signal, and 
thereby alleviating the abnormal conditions. 
The invention also provides a method of treating abnormal conditions 
related to an under-expression of CGRP-RCF activity which comprises 
administering to a subject a therapeutically effective amount of a 
compound which activates the receptor system of the present invention 
(agonists) as described above in combination with a pharmaceutically 
acceptable carrier, to thereby alleviate the abnormal conditions. 
Compositions and Kits 
The soluble form of the CGRP-RCF, and compounds which activate or inhibit 
such polypeptide receptor system, may be employed in combination with a 
suitable pharmaceutical carrier. Such compositions comprise a 
therapeutically effective amount of the polypeptide or compound, and a 
pharmaceutically acceptable carrier or excipient. Such a carrier includes 
but is not limited to saline, buffered saline, dextrose, water, glycerol, 
ethanol, and combinations thereof. The formulation should suit the mode of 
administration. 
The invention also relates to compositions comprising the polynucleotide or 
the polypeptides discussed above. Thus, the polypeptides of the present 
invention may be employed in combination with a non-sterile or sterile 
carrier or carriers for use with cells, tissues or organisms, such as a 
pharmaceutical carrier suitable for administration to a subject. Such 
compositions comprise, for instance, a media additive or a therapeutically 
effective amount of a polypeptide of the invention and a pharmaceutically 
acceptable carrier or excipient. Such carriers may include, but are not 
limited to, saline, buffered saline, dextrose, water, glycerol, ethanol 
and combinations thereof. The formulation should suit the mode of 
administration. 
The invention further relates to pharmaceutical packs and kits comprising 
one or more containers filled with one or more of the ingredients of the 
aforementioned compositions of the invention. 
Administration 
Polypeptides and other compounds of the present invention may be employed 
alone or in conjunction with other compounds, such as therapeutic 
compounds. 
The pharmaceutical compositions may be administered in any effective, 
convenient manner including, for instance, administration by topical, 
oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, 
subcutaneous, intranasal or intradermal routes among others. 
The pharmaceutical compositions generally are administered in an amount 
effective for treatment or prophylaxis of a specific indication or 
indications. In general, the compositions are administered in an amount of 
at least about 10 .mu.g/kg body weight. In most cases they will be 
administered in an amount not in excess of about 8 mg/kg body weight per 
day. Preferably, in most cases, dose is from about 10 .mu.g/kg to about 1 
mg/kg body weight, daily. It will be appreciated that optimum dosage will 
be determined by standard methods for each treatment modality and 
indication, taking into account the indication, its severity, route of 
administration, complicating conditions and the like. 
Gene therapy 
The CGRP-RCF polynucleotides, polypeptides, agonists and antagonists that 
are polypeptides may be employed in accordance with the present invention 
by expression of such polypeptides in vivo, in treatment modalities often 
referred to as "gene therapy." 
Thus, for example, cells from a patient may be engineered with a 
polynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo, and 
the engineered cells then can be provided to a patient to be treated with 
the polypeptide. For example, cells may be engineered ex vivo by the use 
of a retroviral plasmid vector containing RNA encoding a polypeptide of 
the present invention. Such methods are well-known in the art and their 
use in the present invention will be apparent from the teachings herein. 
Similarly, cells may be engineered in vivo for expression of a polypeptide 
in vivo by procedures known in the art. For example, a polynucleotide of 
the invention may be engineered for expression in a replication defective 
retroviral vector, as discussed above. The retroviral expression construct 
then may be isolated and introduced into a packaging cell is transduced 
with a retroviral plasmid vector containing RNA encoding a polypeptide of 
the present invention such that the packaging cell now produces infectious 
viral particles containing the gene of interest. These producer cells may 
be administered to a patient for engineering cells in vivo and expression 
of the polypeptide in vivo. These and other methods for administering a 
polypeptide of the present invention by such method should be apparent to 
those skilled in the art from the teachings of the present invention. 
Retroviruses from which the retroviral plasmid vectors herein above 
mentioned may be derived include, but are not limited to, Moloney Murine 
Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma 
Virus, Harvey Sarcoma Virus, arian leukosis virus, gibbon ape leukemia 
virus, human immunodeficiency virus, adenovirus, Myeloproliferative 
Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral 
plasmid vector is derived from Moloney Murine Leukemia Virus. 
Such vectors well include one or more promoters for expressing the 
polypeptide. Suitable promoters which may be employed include, but are not 
limited to, the retrovirai LTR; the SV40 promoter; and the human 
cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques 
7:980-990 (1989), or any other promoter (e.g., cellular promoters such as 
eukaryotic cellular promoters including, but not limited to, the histone, 
RNA polymerase III, and B-actin promoters). Other viral promoters which 
may be employed include, but are not limited to, adenovirus promoters, 
thymidine kinase (TK) promoters, and B19 parvovirus promoters. The 
selection of a suitable promoter will be apparent to those skilled in the 
art from the teachings contained herein. 
The nucleic acid sequence encoding the polypeptide of the present invention 
will be placed under the control of a suitable promoter. Suitable 
promoters which may be employed include, but are not limited to, 
adenoviral promoters, such as the adenoviral major late promoter; or 
heterologous promoters, such as the cytomegaiovirus (CMV) promoter; the 
respiratory syncytial virus (RSV) promoter; inducible promoters, such as 
the MMT promoter, the metallothionein promoter; heat shock promoters; the 
albumin promoter; the ApoAI promoter; human globin promoters; viral 
thymidine kinase promoters, such as the Herpes Simplex thymidine kinase 
promoter; retroviral LTRs (including the modified retroviral LTRs herein 
above described); the .beta.-actin promoter; and human growth hormone 
promoters. The promoter also may be the native promoter which controls the 
gene encoding the polypeptide. 
The retroviral plasmid vector is employed to transduce packaging cell lines 
to form producer cell lines. Examples of packaging cells which may be 
transfected include, but are not limited to, the PE501, 17, Y-2, Y-AM, 
2, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP+E-86, GP+envAm12, and DAN cell 
lines as described in Miller, A., Human Gene Therapy 1:5-14 (1990). The 
vector may be transduced into the packaging cells through any means known 
in the art. Such means include, but are not limited to, electroporation, 
the use of liposomes, and CaPO4 precipitation. In one alternative, the 
retroviral plasmid vector may be encapsulated into a liposome, or coupled 
to a lipid, and then administered to a host. 
The producer cell line will generate infectious retroviral vector 
particles, which include the nucleic acid sequence(s) encoding the 
polypeptides. Such retroviral vector particles then may be employed to 
transduce eukaryotic cells, either in vitro or in vivo. The transduced 
eukaryotic cells will express the nucleic acid sequence(s) encoding the 
polypeptide. Eukaryotic cells which may be transduced include, but are not 
limited to, embryonic stem cells, embryonic carcinoma cells, as well as 
hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, 
keratinocytes, endothelial cells, and bronchial epithelial cells. 
EXAMPLES 
The present invention is further described by the following examples. The 
examples are provided solely to illustrate the invention by reference to 
specific embodiments. These exemplification's, while illustrating certain 
specific aspects of the invention, do not portray the limitations or 
circumscribe the scope of the disclosed invention. 
Certain terms used herein are explained in the foregoing glossary. 
All examples are carried out using standard techniques, which are well 
known and routine to those of skill in the art, except where otherwise 
described in detail. Routine molecular biology techniques of the following 
examples can be carried out as described in standard laboratory manuals, 
such as Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; 
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), 
herein referred to as "Sambrook." 
All parts or amounts set out in the following examples are by weight, 
unless otherwise specified. 
Unless otherwise stated size separation of fragments in the examples below 
is carried out using standard techniques of agarose and polyacrylamide gel 
electrophoresis ("PAGE") in Sambrook and numerous other references such 
as, for instance, by Goeddel et al., Nucleic Acids Res. 8: 4057 (1980). 
Unless described otherwise, ligations are accomplished using standard 
buffers, incubation temperatures and times, approximately equimolar 
amounts of the DNA fragments to be ligated and approximately 10 units of 
T4 DNA ligase ("ligase") per 0.5 .mu.g of DNA. 
Example 1 
Expression and purification of human CGRP-RCF using bacteria 
The DNA sequence encoding human CGRP-RCF in the deposited polynucleotide is 
digested with specific endonuclease restriction enzyme EcoRI and XhoI to 
release a fragment encoding the open reading frame of CGRP-RCF and the 
fragment is purified and ligated into the bacterial expression vector, 
which is digested with the appropriate enzymes. 
The restrictions sites are convenient to restriction enzyme sites in the 
bacterial expression vectors pQE-9 which are used for bacterial expression 
in these examples. (Qiagen, Inc. Chatsworth, Calif. pQE-9 encodes 
ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin 
of replication ("ori"), an IPTG inducible promoter, a ribosome binding 
site ("RBS"), a 6-His tag and restriction enzyme sites. 
The human CGRP-RCF DNA and the vector pQE-9 both are digested with EcoRI 
and XhoI and the digested DNAs then are ligated together. Insertion of the 
CGRP-RCF DNA into the EcoRI/XhoI restricted vector placed the CGRP-RCF 
coding region downstream of and operably linked to the vector's 
IPTG-inducible promoter and in-frame with an initiating AUG appropriately 
positioned for translation of CGRP-RCF. 
The ligation mixture is transformed into competent E. coli cells using 
standard procedures. Such procedures are described in Sambrook et al., 
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor 
Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain 
M15/rep4, containing multiple copies of the plasmid pREP4, which expresses 
lac repressor and confers kanamycin resistance ("Kan.sup.r "), is used in 
carrying out the illustrative example described here. This strain, which 
is only one of many that are suitable for expressing CGRP-RCF, is 
available commercially from Qiagen. 
Transformants are identified by their ability to grow on LB plates in the 
presence of ampicillin. Plasmid DNA is isolated from resistant colonies 
and the identity of the cloned DNA is confirmed by restriction analysis. 
Clones containing the desired constructs are grown overnight ("O/N") in 
liquid culture in LB media supplemented with both ampicillin (100 ug/ml) 
and kanamycin (25 ug/ml). 
The O/N culture is used to inoculate a large culture, at a dilution of 
approximately 1:100 to 1:250. The cells are grown to an optical density at 
600 nm ("OD.sup.600 ") of between 0.4 and 0.6. 
Isopropyl-B-D-thiogalactopyranoside ("IPTG") is then added to a final 
concentration of 1 mM to induce transcription from lac repressor sensitive 
promoters, by inactivating the lacI repressor. Cells subsequently are 
incubated further for 3 to 4 hours. Cells then are harvested by 
centrifugation and disrupted, by standard methods. Inclusion bodies are 
purified from the disrupted cells using routine collection techniques, and 
protein is solubilized from the inclusion bodies into 8M urea. The 8M urea 
solution containing the solubilized protein is passed over a PD-10 column 
in 2.times. phosphate buffered saline ("PBS"), thereby removing the urea, 
exchanging the buffer and refolding the protein. The protein is purified 
by a further step of chromatography to remove endotoxin. Then, it is 
sterile filtered. The sterile filtered protein preparation is stored in 
2.times. PBS at a concentration of 95 micrograms per mL. 
Example 2 
Cloning and expression of human CGRP-RCF in a baculovirus expression system 
The cDNA sequence encoding the full length human CGRP-RCF protein, in the 
deposited clone is digested with EcoRI/XhoI to release the CGRP-RCF 
fragment. The fragment is then inserted into expression vector, which was 
also digested with the same restriction enzymes. 
The vector pRG1 is used to express the CGRP-RCF protein in the baculovirus 
expression system, using standard methods, such as those described in 
Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL 
CULTURE PROCEDURES, Texas Agricultural Experimental Station Bulletin No. 
1555 (1987). This expression vector contains the strong polyhedrin 
promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) 
followed by convenient restriction sites. The signal peptide of AcMNPV 
gp67, including the N-terminal methionine, is located just upstream of a 
BamH1 site. The polyadenylation site of the simian virus 40 ("SV40") is 
used for efficient polyadenylation. For an easy selection of recombinant 
virus the beta-galactosidase gene from E.coli is inserted in the same 
orientation as the polyhedrin promoter and is followed by the 
polyadenylation signal of the polyhedrin gene. The polyhedrin sequences 
are flanked at both sides by viral sequences for cell-mediated homologous 
recombination with wild-type viral DNA to generate viable virus that 
express the cloned polynucleotide. 
Many other baculovirus vectors could be used in place of pRG1, such as 
pac373, pVL941 and pAcIM1 provided, as those of skill in the an will 
readily appreciate, that construction provides appropriately located 
signals for transcription, translation, trafficking and the like, such as 
an in-frame AUG and a signal peptide, as required. Such vectors are 
described in Luckow et al., Virology 170:31-39, among others. 
The plasmid is digested with the restriction enzymes EcoRI and XhoI and 
then is dephosphorylated using calf intestinal phosphatase, using routine 
procedures known in the art. The DNA is then isolated from a 1% agarose 
gel using a commercially available kit ("Geneclean" BIO 101 Inc., La 
Jolla, Calif.). This vector DNA is designated herein "V2". 
Fragment F2 and the dephosphorylated plasmid V2 are ligated together with 
T4 DNA ligase. E.coli HB101 cells are transformed with ligation mix and 
spread on culture plates. Bacteria are identified that contain the plasmid 
with the human CGRP-RCF gene by digesting DNA from individual colonies 
using EcoRI and XhoI and then analyzing the digestion product by gel 
electrophoresis. The sequence of the cloned fragment is confirmed by DNA 
sequencing. This plasmid is designated herein pBacCGRP-RCF. 
5 .mu.g of the plasmid pBacCGRP-RCF is co-transfected with 1.0 .mu.g of a 
commercially available linearized baculovirus DNA ("BaculoGold.TM. 
baculovirus DNA", Pharmingen, San Diego, Calif.), using the lipofection 
method described by Felgner et al., Proc. Natl. Acad. Sci. USA 
84:7413-7417 (1987). llag of BaculoGold.TM. virus DNA and 5 .mu.g of the 
plasmid pBacCGRP-RCF are mixed in a sterile well of a microtiter plate 
containing 50 .mu.l of serum free Grace's medium (Life Technologies Inc., 
Gaithersburg, Md.). Afterwards 10 .mu.l Lipofectin plus 90 .mu.l Grace's 
medium are added, mixed and incubated for 15 minutes at room temperature. 
Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC 
CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium 
without serum. The plate is rocked back and forth to mix the newly added 
solution. The plate is then incubated for 5 hours at 27.degree. C. After 5 
hours the transfection solution is removed from the plate and 1 ml of 
Grace's insect medium supplemented with 10% fetal calf serum is added. The 
plate is put back into an incubator and cultivation is continued at 
27.degree. C. for four days. 
After four days the supernatant is collected and a plaque assay is 
performed, as described by Summers and Smith, cited above. An agarose gel 
with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow 
easy identification and isolation of gal-expressing clones, which produce 
blue-stained plaques. (A detailed description of a "plaque assay" of this 
type can also be found in the user's guide for insect cell culture and 
baculovirology distributed by Life Technologies Inc., Gaithersburg, page 
9-10). 
Four days after serial dilution, the virus is added to the cells. After 
appropriate incubation, blue stained plaques are picked with the tip of an 
Eppendorf pipette. The agar containing the recombinant viruses is then 
resuspended in an Eppendorf tube containing 200 .mu.l of Grace's medium. 
The agar is removed by a brief centrifugation and the supernatant 
containing the recombinant baculovirus is used to infect Sf9 cells seeded 
in 35 mm dishes. Four days later the supernatants of these culture dishes 
are harvested and then they are stored at 4.degree. C. A clone containing 
properly inserted CGRP-RCF is identified by DNA analysis including 
restriction mapping and sequencing. This is designated herein as 
V-CGRP-RCF. 
Sf9 cells are grown in Grace's medium supplemented with 10% 
heat-inactivated FBS. The cells are infected with the recombinant 
baculovirus V-CGRP-RCF at a multiplicity of infection ("MOI") of about 2 
(about 1 to about 3). Six hours later the medium is removed and is 
replaced with SF900 II medium minus methionine and cysteine (available 
from Life Technologies Inc., Gaithersburg). 42 hours later, 5 .mu.Ci of 
35S-methionine and 5 .mu.Ci 35S cysteine (available from Amersham) are 
added. The cells are further incubated for 16 hours and then they are 
harvested by centrifugation, lysed and the labeled proteins are visualized 
by SDS-PAGE and autoradiography. 
Example 3 
Gene therapeutic expression of human CGRP-RCF 
Fibroblasts are obtained from a subject by skin biopsy. The resulting 
tissue is placed in tissue-culture medium and separated into small pieces. 
Small chunks of the tissue are placed on a wet surface of a tissue culture 
flask, approximately ten pieces are placed in each flask. The flask is 
turned upside down, closed tight and left at room temperature overnight. 
After 24 hours at room temperature, the flask is inverted--the chunks of 
tissue remain fixed to the bottom of the flask--and fresh media is added 
(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin). The 
tissue is then incubated at 37.degree. C. for approximately one week. At 
this time, fresh media is added and subsequently changed every several 
days. After an additional two weeks in culture, a monolayer of fibroblasts 
emerges. The monolayer is trypsinized and scaled into larger flasks. 
A vector for gene therapy is digested with restriction enzymes for cloning 
a fragment to be expressed. The digested vector is treated with calf 
intestinal phosphatase to prevent self-ligation. The dephosphorylated, 
linear vector is fractionated on an agarose gel and purified. 
CGRP-RCF cDNA capable of expressing active CGRP-RCF, is isolated. The ends 
of the fragment are modified, if necessary, for cloning into the vector. 
For instance, 5' overhanging may be treated with DNA polymerase to create 
blunt ends. 3' overhanging ends may be removed using S1 nuclease. Linkers 
may be ligated to blunt ends with T4 DNA ligase. 
Equal quantities of the Moloney murine leukemia virus linear backbone and 
the CGRP-RCF fragment are mixed together and joined using T4 DNA ligase. 
The ligation mixture is used to transform E. coli and the bacteria are 
then plated onto agar-containing kanamycin. Kanamycin phenotype and 
restriction analysis confirm that the vector has the properly inserted 
gene. 
Packaging cells are grown in tissue culture to confluent density in 
Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), 
penicillin and streptomycin. The vector containing the CGRP-RCF gene is 
introduced into the packaging cells by standard techniques. Infectious 
viral particles containing the CGRP-RCF gene are collected from the 
packaging cells, which now are called producer cells. 
Fresh media is added to the producer cells, and after an appropriate 
incubation period media is harvested from the plates of confluent producer 
cells. The media, containing the infectious viral particles, is filtered 
through a Millipore filter to remove detached producer cells. The filtered 
media then is used to infect fibroblast cells. Media is removed from a 
sub-confluent plate of fibroblasts and quickly replaced with the filtered 
media. Polybrene (Aldrich) may be included in the media to facilitate 
transduction. After appropriate incubation, the media is removed and 
replaced with fresh media. If the liter of virus is high, then virtually 
all fibroblasts will be infected and no selection is required. If the 
titer is low, then it is necessary to use a retroviral vector that has a 
selectable marker, such as neo or his, to select out transduced cells for 
expansion. 
Engineered fibroblasts then may be injected into rats, either alone or 
after having been grown to confluence on microcarrier beads, such as 
cytodex 3 beads. The injected fibroblasts produce CGRP-RCF product, and 
the biological actions of the protein are conveyed to the host. 
It will be clear that the invention may be practiced otherwise than as 
particularly described in the foregoing description and examples. 
Numerous modifications and variations of the present invention are possible 
in light of the above teachings and, therefore, are within the scope of 
the appended claims. 
Example 4 
In Vitro Transcription, Microinjection into Xenopus laevis oocytes and 
Electrophysiology 
Capped RNA transcripts were synthesized from linearized pCGRP-RCF or 
pCGRP-type-I-receptor DNA using T7 RNA polymerase (Stratagene). For 
microinjection, X. laevis oocytes were prepared as previously described 
(Elshourbagy et al., J. Biol. Chem., 268:3873-3879 (1993)). Stage V-VI 
oocytes were selected, and the follicular membranes were manually removed. 
For each experimental group, 6 defolliculated oocytes were coinjected with 
50 nl of water containing 20 ng of CGRP-RCF and CGRP type I receptor 
complementary RNA (cRNA) (Drummond injection apparatus). The injected 
oocytes were maintained in modified Barth's medium at 18.degree. C. for 48 
hrs to allow for protein synthesis complex to the cell surface. 
Electrophysiology was performed using the voltage clamp technique using an 
oocyte voltage clamp apparatus (Warner Instruments). Oocytes were clamped 
at -60 mV, and exposed to 10.sup.-7 M human alpha-CGRP or human Calcitonin 
(Bachere) and the Ca.sup.+2 activated channel activity was recorded in 
Barth's medium at room temperature. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1450 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: 
(vi) ORIGINAL SOURCE: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GGCACGAGCAGCTGTGAAGTGTGAGGTTCTTTGTCTGCTGGCAGCTAGGGGCGACGAGGC60 
GGGACGTCATGGAAGTGAAGGATGCCAATTCTGCGCTTCTCAGTAACTACGAGGTATTTC120 
AGTTACTAACTGATCTGAAAGAGCAGCGTAAAGAAAGTGGAAAGAATAAACACAGCTCTG180 
GGCAACAGAACTTGAACACTATCACCTATGAAACGTTAAAATACATATCAAAAACACCAT240 
GCAGGCACCAGAGTCCTGAAATTGTCAGAGAATTTCTCACAGCATTGAAAAGCCACAAGT300 
TGACCAAAGCTGAGAAGCTCCAGCTGCTGAACCACCGGCCTGTGACTGCTGTGGAGATCC360 
AGCTGATGGTGGAAGAGAGTGAAGAGCGGCTCACGGAGGAGCAGATTGAAGCTCTTCTCC420 
ACACCGTCACCAGCATTCTGCCTGCAGAGCCAGAGGCTGAGCAGAAGAAGAATACAAACA480 
GCAATGTGGCAATGGACGAAGAGGACCCAGCATAGAAGAGCACAGCTGGCCCCGGCGTTT540 
CATGAAGTCAGAAGGCCTGGCAGCCATTTCCTGGACGTTGAGAGGATTGTTTATTTGATT600 
TTTATCCTCATCCCAGCAGGCCTGGCTTTGTGGTTAGTTGGGTACATCACAAAAATAAGT660 
TAAAAAGAAATATTTGTGCCTTGGGGAGAAGAAACATGGTGAAAACAGGCTGAGGTTGTC720 
AGGGCAGAGAGCTGAAGGTGGGGACAGTGACCGCGGACCCCTCTGCGCTTGAAAGATTTC780 
CTCCACGGCCTTTGCCCCAGTTGTGGGGAGGTCTCTGTGCACAGCGGGGAAAATGCTTGT840 
GTCGCCTTTGGTGGGCCATGTCCTAATTAGTTTCATCTGCTTCCCTGGGAACTTACTAAG900 
GGGCCCAGAGCACTGTTGGAAGTCTGGTTAGAGTCCCCAGAGAGTTACTCTAAGTTAAAA960 
TGAGCCACTGACCTTGGCTCACCTTAGAGGAATTTCCTCGAGAACAACAGAGATAAGAAA1020 
AGAACCGGCCTGGCCAATCCTTCAACAGCTCTAGAGCCCCTTTTCTCTGCTGGCAGGGGC1080 
TTTGTTTACCAGCTCACTGTTTAGGCTAAATGTTAGGGACCAGATCACTGCAGTTGAAAA1140 
CAGCATCCAGGCTTAGTGACAGTGGCAGCAGAAACAGTGTTGGCTGCCTTTCTGACCACC1200 
CCACTTTCCTGCCCTGAGACAGCAGCCCAGGGCAGGTGCTTCATATTCAGACCAGGTAAG1260 
CCTCATTTGCACAACAGTCAAATTGTTTGTTCCTTTAAAAAGGACACAATTAGCCTGGCA1320 
CGGTGACTCATGCTTGTAATCCCAGCACTTTGGGAGGGCAAGGCAGGCGGATCACCTGAG1380 
GTCAGGAGTTTGAGACCAGCCTCACCAACATGGAAAAACCCCATCTCTATTAAAAAAAAA1440 
AAAAAAAAAA1450 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 148 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: N-terminal 
(vi) ORIGINAL SOURCE: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetGluValLysAspAlaAsnSerAlaLeuLeuSerAsnTyrGluVal 
151015 
PheGlnLeuLeuThrAspLeuLysGluGlnArgLysGluSerGlyLys 
202530 
AsnLysHisSerSerGlyGlnGlnAsnLeuAsnThrIleThrTyrGlu 
354045 
ThrLeuLysTyrIleSerLysThrProCysArgHisGlnSerProGlu 
505560 
IleValArgGluPheLeuThrAlaLeuLysSerHisLysLeuThrLys 
65707580 
AlaGluLysLeuGlnLeuLeuAsnHisArgProValThrAlaValGlu 
859095 
IleGlnLeuMetValGluGluSerGluGluArgLeuThrGluGluGln 
100105110 
IleGluAlaLeuLeuHisThrValThrSerIleLeuProAlaGluPro 
115120125 
GluAlaGluGlnLysLysAsnThrAsnSerAsnValAlaMetAspGlu 
130135140 
GluAspProAla 
145 
__________________________________________________________________________