WO1997041223A1 - Clone moleculaire de proteine composante du recepteur du cgrp et son utilisation - Google Patents

Clone moleculaire de proteine composante du recepteur du cgrp et son utilisation Download PDF

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WO1997041223A1
WO1997041223A1 PCT/US1997/006321 US9706321W WO9741223A1 WO 1997041223 A1 WO1997041223 A1 WO 1997041223A1 US 9706321 W US9706321 W US 9706321W WO 9741223 A1 WO9741223 A1 WO 9741223A1
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cgrp
rcp
protein
nucleic acid
subject
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PCT/US1997/006321
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English (en)
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Ian Dickerson
Anne Luebke
Gerhard Dahl
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The University Of Miami
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Priority to AU27317/97A priority Critical patent/AU2731797A/en
Publication of WO1997041223A1 publication Critical patent/WO1997041223A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of neuropeptide receptors. More specifically, the invention relates to the cloning of the calcitonin gene related peptide (CGRP) receptor component protein (CGRP-RCP) and the use of the clone to develop diagnostic assays for CGRP-RCP and to screen for agonists and antagonists of CGRP.
  • CGRP calcitonin gene related peptide
  • Calcitonin gene-related peptide is a 37 amino-acid carboxyl-amidated neuropeptide secreted by nerves of the central and peripheral nervous systems (Amara, S .G . et al. (1982) Nature. 298:240-244; and Rosenfeld, M.G. et al. (1983) Nature, 304:129-135. ) .
  • CGRP one of the most potent vasodilators known (Brain, S.D. et al. (1985) Nature. 313:54-56), may also be a neuromodulator (Vetter, D.E. et al. (1991) Svnaose.
  • CGRP has a plethora of functions in the body. Aside from its vasodilatory and neuromodulatory actions, CGRP has been proposed to increase acetylcholine receptor synthesis at the neuromuscular junction (Oesterlund, M. et al. (1989) Neuroscience. 32:279-287), and to desensitize the acetylcholine receptor at the neuromuscular junction and the efferent synapses of the inner ear (Wackym, P.A. et al.
  • CGRP-containing efferent fibers from the brainstem synapse at the inner ear end-organ sites, perhaps contributing to the processes responsible for the detection of auditory signals in the presence of background noise.
  • CGRP has been implicated as a growth factor for human endothelial cells (Haegerstrand, A.
  • Protein purification strategies based on cross- linking cell extracts to CGRP followed by analysis by SDS- PAGE have identified candidate proteins for the CGRP receptor and its complex, with molecular weights ranging from 17-70 kD (Hirata, Y. et al . (1988) . Biochem. Biophys. Res. Commun.. 151:1113-1121; Sano, Y. et al. (1989) . J_ ⁇ Neurochem.. 52:1919-1924; Stangle, D. et al . (1991) . Biochem.. 30:8605-8611; Chatterjee, T.K. et al . (1993) . Mol . Pharmacol ..
  • the present invention relates to the cloning of a calcitonin gene - related peptide receptor component protein (CGRP-RCP) .
  • CGRP-RCP calcitonin gene - related peptide receptor component protein
  • the present invention therefore, provides purified and isolated nucleic acid molecules encoding the CGRP-RCP.
  • the invention also relates to the use of single- stranded antisense poly-or oligonucleotides derived from the CGRP-RCP nucleic acid molecules to inhibit the expression of CGRP-RCP.
  • nucleic acid molecule refers to RNA, DNA, cDNA, cRNA or any synthetic variant thereof.
  • the present invention also provides vectors containing the nucleic acid molecules, a host cell stably transformed or transfected with such vectors, as well as the CGRP-RCP produced by the transformed host cell. The present invention therefore relates to the CGRP-RCP and peptide fragments thereof.
  • the present invention also provides methods for detecting nucleic acid encoding CGRP-RCP in a biological sample.
  • the method comprises the steps of: (a) contacting nucleic acid from the biological sample with probes made from nucleic acid encoding the CGRP-RCP protein under conditions permitting a complex to be formed between the probe and nucleic acid present in the sample; and (b) detecting the formation of the complex.
  • the method comprises the steps of: (a) contacting nucleic acid from the biological sample with sense and antisense primers prepared from nucleic acid encoding the CGRP-RCP under conditions permitting PCR amplification to occur; and (b) detecting amplification of the nucleic acid from the biological sample.
  • the present invention also provides a method for detecting CGRP-RCP in a biological sample which comprises the steps of: (a) contacting the biological sample with antibody which specifically reacts with CGRP-RCP; and
  • the invention therefore also relates to antibodies directed to the CGRP-RCP or to peptide fragments of CGRP- RCP.
  • the present invention also provides methods for identifying ligands capable of binding to the CGRP receptor.
  • the method comprises the steps of:
  • the present invention further provides a method for screening for drugs capable of acting as agonists or antagonists of CGRP which comprises the steps of: (a) transfecting Xenopus oocytes with nucleic acid molecules encoding CGRP-RCP; (b) contacting the transformed oocytes with candidate drugs; and (b) evaluating the biological activity mediated by contact with the candidate drugs.
  • the present invention therefore provides the agonists and antagonists identified by the above methods as well as pharmaceutical compositions comprising these agonists or antagonists and the use of these compositions in therapeutic and/or prophylactic methods.
  • the present invention provides a method for treating hypertension in a subject comprising administering to the subject an amount of an agonist of CGRP effective to lower blood pressure.
  • the present invention also provides a method for treating glaucoma in a subject comprising administering to the subject a therapeutically effective amount of an antagonist to CGRP.
  • the present invention also provides a method for treating premature labor in a subject comprising administering to the subject a therapeutically effective amount of an agonist of CGRP.
  • Figure 1 is a schematic which illustrates the oocyte-CFTR assay.
  • Peptide binds to receptor, resulting in activation of G,.
  • G s stimulates adenylate cyclase, which activates protein kinase A (PKA) .
  • PKA protein kinase A
  • PKA phosphorylates the cystic fibrosis transmembrane conductance regulator (CFTR) , resulting in an inward current.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Figures 2A and 2B show CFTR Cl " current from (A) a voltage-clamped oocyte injected with 20 ng CFTR cRNA and challenged with 20 ⁇ M forskolin and (B) from successive pools of clones (The number of species per pool is (l) 100,000, (2) 5,000, (3) 625 (4) 78, (5) 10, (6) 1.) Injected oocytes were incubated for 24 hrs before recording. The membrane potential of oocytes was clamped at -50 rriV for all experiments.
  • inward currents are depicted as an upward deflection in the current trace; upward arrows indicate beginning of application of reagents and downward arrows indicate beginning of reagent washout.
  • Figures 3A and 3B show CFTR Cl " currents from A) an individual oocyte coinjected with 20 ng CFTR cRNA and 20 ng CGRP receptor cRNA and challenged with increasing levels of CGRP ( IO '9 M, 5xl0 "9 M,10 "8 M, 5xl0 “8 M, IO “7 M) and B) from eight pools (3-6 oocytes/pool) coinjected with 20 ng CFTR cRNA and 20 ng CGRP-RCP cRNA, incubated for 24 hrs and then challenged with CGRP at the indicated concentrations.
  • the inset of Figure 3A shows the first three points plotted with an expanded scale.
  • the open square indicates the lack of currents obtained upon application of IO '7 M of any other peptide tested.
  • inward currents are depicted as an upward deflection in the current trace, upward arrows indicate beginning of application of reagents and downward arrows indicate beginning of reagent washout.
  • Figures 4A and 4B show nucleotide sequence and predicted amino acid sequence for the guinea pig cochlear CGRP-RCP cDNA ( Figure 4A) and a Kyte-Doolittle hydrophilicity plot of the deduced amino acid sequence for the cochlear CGRP receptor ( Figure 4B) .
  • single underlined sequences 1 and 2 represent the two oligonucleotide primers used for PCR on cerebellar mRNA (see Figure 2B) ; double-underlined sequence 3 represents an antisense oligonucleotide used for experiments shown in Figure 5; single underlined sequence 4 represents primer used for direct testing of ORF in Figure 2B; and Kozak translational initiation consensus sequence is indicated by a bar over the sequence.
  • Figure 5 shows a Northern blot of guinea pig cerebellar RNA where the lane was loaded with 20 ⁇ g of cerebellar total RNA and probed with a full-length random- primed CGRP-RCP PCR product. Cerebellar CGRP-RCP RNA migrated with an estimated size of 1.8 kb. Positions of guinea pig rRNA bands are indicated on the right of the Figure.
  • Figure 6 shows in vitro translation of CGRP-RCP cRNA translated in vitro with canine microsomal pancreatic extracts (lane 1) , of CGRP-RCP cRNA translated in vitro without microsomal extracts (lane 2) , and control in vitro translation with no cRNA added (lane 3) .
  • Figures 7A-7C show CFTR Cl " currents from A) an oocyte injected with 20 ng of guinea pig cerebellar mRNA and 20 ng CFTR cRNA; B) from an oocyte injected with 20 ng of antisense CGRP-RCP oligonucleotide in addition to guinea pig cerebellar mRNA and CFTR cRNA; and C) from oocytes coinjected with CFTR cRNA (20 ng) and cRNA from either CGRP- RCP cDNA (200 pg, solid bar) or cerebellar mRNA (20 ng, striped bar) , with or without antisense CGRP-RCP (20 ng) oligonucleotide (AS) and incubated with 10 "7 M CGRP.
  • Figure 8 shows two photomicrographs depicting the results of immunocytochemistry using an antibody to CGRP on a microdissected basal turn of the guinea pig organ of Corti.
  • the top panel shows immunostaining of CGRP-RCP- containing efferent fiber terminals on the three rows of outer hair cells (OHCs, labeled 1, 2, and 3 respectively) .
  • the bottom panel shows no staining when the pre-immune serum was substituted for the CGRP-RCP antibody.
  • Figure 9 shows two photomicrographs depicting the results of in situ hybridization using an antisense riboprobe to the CGRP-RCP on a microdissected basal turn of the guinea pig organ of Corti.
  • the top panel shows the staining of the three rows of outer hair cells (OHCs labeled 1, 2 and 3 respectively) with the digoxigenin-labeled antisense probe, and the bottom panel shows background staining observed using the digoxigenin-labeled sense probe.
  • Figure 10 shows the alignments between the deduced amino acid sequences of the human, guinea pig, rabbit and mouse CGRP-RCP cDNA clones and the partial chick and rat CGRP-RCP PCR amplimers.
  • the single letter abbreviation for the amino acids are those known in the art.
  • the solid black residues match the guinea pig CGRP-RCP sequence.
  • the hyphens (-) indicate gaps introduced by the alignment software to maximize the amino acid alignment of the cDNA clones.
  • Figures 11 A and 11 B shows the alignments between the coding sequences of CGRP-RCP cDNAs (human, guinea pig, rabbit and mouse) and PCR amplimers (chick and rat) .
  • the solid black residues match the guinea pig CGRP-RCP sequence.
  • the hyphens (-) indicate gaps introduced by the alignment software to maximize the nucleic acid alignment of the cDNA clones.
  • Figure 12 shows the map of the mouse CGRP-RCP gene. Sizes of known exons and introns are shown above the map. Exons are marked as open boxes, introns as single lines; RNA splicing Donor (D) and Acceptor (A) sites are marked. Donor Dl and acceptor Al are used to splice the cochlear form of the CGRP-RCP; D2 and Al are used for the alternatively spliced form found in the cerebellum. Start codon (ATG) and stop codons (TAG) are shown.
  • Figures 13A and 13B show records of ACh-induced contractions of muscle strips isolated from mouse myometrium (longitudinal layer) at various stages of pregnancy. In all records the strips were induced to contract by incubation with lO ⁇ M ACh alone (first and third contraction) or by lO ⁇ M ACh and 0.l ⁇ M- CGRP after a 1 minute preincubation with O.l ⁇ M CGRP (second contraction) .
  • Figure 14 shows the nucleotide sequence of mouse CGRP-RCP.
  • Arrows 1, 2 and 3 represent the position of the degenerate oligonucleotide primers DRES-l, DRES-2 and DRES-3, used for RT-PCR on uterine mRNA.
  • Primers MRES-5B and MRES-6B were used for 3' and 5' RACE, respectively.
  • Primers T7-M1 and MRES-17 were used for PCR amplification of the CGRP-RCP ORF. Amino acid sequence used to raise CGRP-RCP antibody is underlined.
  • Figure 15 shows the amino acid sequence alignment of guinea pig and mouse CGRP-RCP, using the program Megalign (DNA STAR, Inc.). Mouse and guinea pig CGRP-RCP are 86% identical at the amino acid level.
  • Figure 16 shows the in vi tro translation of the mouse CGRP-RCP (arrow) in the presence of 35 S methionine and resolved by 15% SDS-PAGE. Molecular weight markers are indicated.
  • FIGS 17A and 17B show the results of a functional analysis of the mouse CGRP-RCP using the oocyte-CFTR assay. Shown are the membrane currents (CFTR Cl- currents) induced by application of 1 OOnM CGRP to Xenopus oocytes injected with CFTR cRNA and either CGRPRCP ORF cRNA
  • Figure 17A or myometrial mRNA alone (Figure 17B) or with myometrial mRNA mixed with an antisense oligonucleotide made to the CGRP-RCP cDNA sequence ( Figure 17B) .
  • the membrane potential of the oocytes was clamped at -50mV.
  • Inward currents are depicted as an upward deflection. Upward arrows indicate addition of ligand, downward arrows indicate washout.
  • Figure 18A shows Northern blot analysis of mouse uterine CGRP-RCP mRNA during gestation (lanes 1-11: embryonic days 6 through 18) , at parturition (lane 12) , and postpartum (lanes 13-14: postnatal day 1 and 2) .
  • RNA 2 ⁇ g/lane was hybridized to a 32 P-labeled CGRP-RCP cDNA probe. Positions of 18S and 28S ribosomal RNA are indicated by arrows.
  • Figure 18B shows quantitative analysis of uterine mRNA levels determined by densitometrie scanning of three independent northern blots. For analysis, the membranes were stripped after the first hybridization and rehybridized to a GAPDH probe (not shown) , and the CGRP-RCP signal was normalized to the GAPDH signal. Normalized CGRP-RCP mRNA is expressed as a fraction of the mRNA level at day E6 to account for variations between autoradiographic films. The bars represent the means ⁇ SEM of 3 measurements.
  • Figures 19A-19C show the correlation of CGRP-RCP protein expression with CGRP inhibition of ACh induced contraction during gestation.
  • Figure 19A shows Western blot analysis of mouse uterine tissue with CGRP-RCP antibody. Solubilized protein extracts (75 ⁇ g/lane) from uteri of pregnant mice (lanes 1-11: E6parturition) and postpartum mice (lanes 12 and 13: PN1 and PN2) were separated by 15% SDS-PAGE, transferred to Immobilon membrane, and immunoblotted with CGRP-RCP antibody.
  • Figure 19B shows quantitative analysis of uterine
  • CGRP-RCP protein levels determined by densitometric scanning of the 20kDa bands from three western blots. Values are expressed as a fraction of the signal intensity obtained from E6 uterus. Means ⁇ SEM of 3 measurements are given.
  • Figure 19C shows quantitative analysis of CGRP inhibition of ACh-induced contractions of uterine smooth muscle preparations from gestating and postpartum mice. Bars represent percent inhibition produced by incubation with
  • Figures 20A-20C show the alignments between the coding sequences of the two alternately spliced forms of the CGRP-RCP cDNA.
  • Sequences denoted by ORF.Seq correspond to cDNAs first isolated from guinea pig cochlear RNA and are generated by splicing pre-mRNA from Dl to Al (see Figure 12).
  • Sequences denoted by ORF2.Seq correspond to cDNAs observed only in the cerebellum, and arise by splicing of pre-mRNA from D2 to Al (see Figure 12) .
  • the hyphens (-) indicate gaps introduced by the alignment software to maximize the alignment of the cDNA sequences.
  • Figure 20A shows guinea pig sequences
  • Figure 20B shows human sequences
  • Figure 20C shows mouse sequences.
  • the present invention relates to the cloning of a calcitonin gene-related peptide receptor component protein (CGRP-RCP) using an expression cloning strategy in which the cystic fibrosis transmembrane conductance regulator (CFTR) , a protein kinase A activated chloride channel, was used as a sensor for CGRP-induced activation of intracellular kinases. It is believed that the CGRP-RCP is either the CGRP receptor or a member of a complex of proteins that together constitute the CGRP receptor.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the present invention therefore provides a purified and isolated nucleic acid molecule encoding CGRP- RCP.
  • the nucleic acid molecule includes all nucleic acid sequences which encode for the CGRP-RCP including all degenerate forms.
  • the invention provides a full-length cDNA for guinea pig CGRP-RCP protein. This cDNA sequence is set forth below as SEQ ID NO:l
  • AGCGCCCCAT TTCCTGTCCT GAGCCAGCCC CTGGTTGGGC 1080
  • AAAAAAAAAA AAAAAA 1736 where the abbreviations used for the nucleotides are those standardly used in the art.
  • CGRP-RCP cDNA is shown below as SEQ ID NO:2 starting at nucleotide 49 of SEQ. ID. NO:l:
  • the present invention provide a cDNA sequence encoding the open-reading frame for the human CGRP-RCP. This cDNA sequence is shown below as SEQ NO:3
  • SEQ ID NO:3 is shown below as SEQ ID NO:4. and starts at nucleotide i of SEQ. ID. NO:3.
  • the nucleic acid sequences of the invention comprise the nucleotide sequence shown in SEQ ID NO:3, or a sequence which is substantially homologous thereto.
  • substantially homologous is meant that the level of identity between SEQ ID NO:3 and the corresponding portion of the nucleic acid sequence of interest is at least 50%, preferably about 65%, and most preferably, about 70-90%.
  • homologoues of the guinea pig and human CGRP-RCP cDNAs provided herein may be isolated from other species using the nucleic acids shown in SEQ ID NO: 1 and 3, or portions thereof, as probes or primers. Indeed, using RT-PCR, cDNA clones from mouse and rabbit as well as from guinea pig and human, have been obtained and sequenced (see Figures HA and HB) . In addition, degenerate primers designed to regions of guinea pig CGRP- RCP were utilized to carry out degenerate PCR on mRNA isolated from rat and chick and the resulting sequences of the PCR amplimers are shown in Figure 11. The % identity of the human CGRP-RCP nucleotide and amino acid sequences with each of the CGRP-RCP nucleotide and amino acid sequences obtained from the other species was determined using Wilbur- Lipman paradigm and are shown below:
  • calcitonin receptors are an example of neuropeptide receptors which exist as a family of subtypes; the calcitonin receptor family consisting of core cDNA into which can be added, by use of alternative exons, several extra exons to derive the 4 subtypes described to date.
  • calcitonin receptors are an example of neuropeptide receptors which exist as a family of subtypes; the calcitonin receptor family consisting of core cDNA into which can be added, by use of alternative exons, several extra exons to derive the 4 subtypes described to date.
  • nucleic acid sequences of the invention may be used as probes to screen libraries of cDNA clones for additional members of the CGRP-RCP family using standard DNA hybridization techniques. It is expected that DNA sequences encoding the different members of the CGRP-RCP family will exhibit a level of similarity that clearly exceeds the 50% identity required for cross-hybridization between homolog of CGRP-RCP from different species.
  • sequences of the present invention may also be used as probes to isolate genomic clones encoding the CGRP-RCP family by screening human genomic libraries using standard DNA hybridization techniques.
  • the mouse gene has been cloned using probes derived from the mouse CGRP-RCP cDNA ( Figures HA and HB) and a map of the gene is shown in Figure 12.
  • the mouse CGRP-RCP gene has been localized to mouse chromosome 5 between 78 and 79 centimorgans according to Mouse Genome Database on the Internet.
  • the present invention therefore relates to the use of the mouse gene to generate "knock-out" mice with a defect in the gene encoding the mouse counterpart of the human CGRP-RCP.
  • knock-out mice may be generated by techniques known to those or ordinary skill in the art and would be extremely valuable tools to further explore the role of CGRP-RCP in the multiple functions of CGRP in the body.
  • oligonucleotide primers that span an intron could be used to screen mRNA selectively, while primers that are either contained within introns or span intron/exon junction could be used to selectively screen genomic DNA.
  • the present invention also provides methods for detecting nucleic acids encoding CGRP-RCP in a biological sample utilizing as probes the CGRP-RCP nucleic acid sequences shown herein, or portions thereof, labeled with detectable markers.
  • biological samples examples include, but are not limited to, animal tissues such as cerebellum, cerebrum, heart, liver, lung, skeletal muscle and tongue, cell samples and eukaryotic or prokaryotic cells.
  • the method comprises the steps of: (a) contacting the probe with nucleic acids from a biological sample under conditions permitting a complex to be formed between the probe and nucleic acid present in the sample; and (b) detecting the formation of the complex.
  • the nucleic acid may be extracted from the biological samples by known procedures. Specifically, the cells may be lysed using an enzyme such as proteinase K, in the presence of detergents such as sodium dodecyl sulfate (SDS), NP40, or Tween 20. If the nucleic acid is genomic DNA, it may be extracted using known techniques such as phenol/chloroform extraction. Alternatively, the DNA may be isolated using one of the commercially available kits such as the Oncor Genomic DNA isolation kit. If the nucleic acid to be isolated is RNA, the RNA to be extracted may be whole cell RNA or poly A+RNA. Whole cell RNA can be isolated by methods known to those skilled in the art. Such methods include extraction of RNA by different precipitation
  • RNA can be selected from whole cell RNA by affinity chromatography on oligo-d(T) columns (Aviv, H. et al . (1972) Proc. Natl. Acad. Sci., 69:1408-1412) .
  • the formation of the complex may be detected using various conventional techniques known in the art, including, but not limited to, Northern blotting, Southern blotting, dot and slot hybridization, SI nuclease assay, ribonuclease protection assay, and filter hybridization (Southern, E.M. J. Mol. Biol.. 98:503 (1975) ; Chirgwin, J.M. , et al . Biochemistry. 18:5294-5299 (1979); Kafatos, et al. Nuc. Acids. Res.. 7:1541 (1979); Thomas, P.S. Proc. Natl. Acad. Sci.. 77:5201 (1980) ; White, B.A. and F.C.
  • the probe is labeled with a detectable marker by techniques known to those or ordinary skill in the art.
  • labelling techniques can include enzymes and radiolabels such as 32 P (Sambrook, J. et al . (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview NY) .
  • radiolabels such as 32 P (Sambrook, J. et al . (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview NY) .
  • non-radioactive techniques for signal amplification including methods for attaching chemical moieties to pyrimidine and purine rings (Dale, R.N.K. et al. (1973) Proc. Natl. Acad. Sci.. 70:2238-2242; Heck, R.F. (1968) S_ ⁇ Am. Chem. Soc..
  • the marker is 32 P and the probe is labeled by known procedures such as random priming (Sambrook, J., Fritsch, E.F., and Maniatis, T. "Molecular Cloning, A Laboratory Manual,” Second Edition, Cold Spring Harbor Laboratory Press, pp. 11.31-11.32 (1989) ) .
  • probes of the present invention may be designed to detect all members of the CGRP- RCP family or they may be unique in detecting only one or more specific members of the CGRP-RCP family.
  • the present invention also provides a method for detecting nucleic acid encoding CGRP-RCP in a biological sample using standard PCR procedures.
  • the method comprises the steps of: (a) contacting nucleic acid from the biological sample with a sense and an antisense primer prepared from the sequence of the present invention under conditions permitting PCR amplification to occur; and (b) detecting amplification of the nucleic acid from the biological sample.
  • Polymerase chain reaction is performed by methods and conditions disclosed in U.S. Patent Nos. 4,683,202 and 4,683,195 and in Perkin Elmer Cetus PCR kit protocols.
  • the DNA polymerase, deoxyribonucleotide triphosphates (dNTPS) (e.g. dATP, dCTP, dTTP, and dGTP), and amplification bu f fer (e.g. glycerol, tri-hydrochloric acid, potassium chloride, Tween 20, and magnesium chloride) are readily commercial l y available (Perkin Elmer Cetus) .
  • the polymerase chain reaction may be performed for as many cycles as desired.
  • RT-PCR Reverse transcription-PCR
  • mRNA is by methods described in commercially available kits such as the RT-PCR kit of Perkin Elmer Cetus.
  • the sense and antisense primers for use in PCR and RT-PCR may be prepared from the CGRP-RCP nucleic acid sequences provided herein using automated instruments sold by a variety of manufacturers or they may be commercially synthesized.
  • the primers can be derivatized to include a detectable label suitable for detecting and/or identifying the primer extension products (eg. radiolabelled dNTPs) or with a substance that aids in isolation of the products of amplification.
  • a detectable label suitable for detecting and/or identifying the primer extension products (eg. radiolabelled dNTPs) or with a substance that aids in isolation of the products of amplification.
  • the amplification products of PCR can be detected directly via the use of labeled primer pairs.
  • Labels suitable for labelling primers include radioactive labels, biotin, avidin, enzymes and fluorescent molecules.
  • the desired label can be incorporated into the primer extension products during the amplification reaction in the form of one or more labelled dNTPs.
  • unlabelled amplification products can be detected by hybridization with labelled nucleic acid probes derived from nucleic acid molecules encoding CGRP-RCP. It is within the confines of the present invention that primers for PCR amplification may be designed to detect all members of the CGRP-RCP family, or may be unique in detecting only one or more specific members of the CGRP-RCP family. The present invention also provides recombinant
  • CGRP receptor component proteins produced by recombinant DNA methodologies known to those of ordinary skill in the art using vectors comprising nucleic acid molecules encoding CGRP-RCP.
  • Suitable vectors for use in the invention are those that function in prokaryotic or eukaryotic cell. By suitable is meant that the vector is capable of carrying and expressing a nucleic acid sequence encoding a CGRP receptor component protein.
  • Vectors contemplated for use in the invention therefore include any vector into which a nucleic acid sequence encoding CGRP-RCP can be inserted, along with any preferred or required expression control elements, and which vector can subsequently be transferred into a prokaryotic or eukaryotic host cell.
  • Preferred expression vectors are those that function is eukaryotic host cells.
  • vectors examples include, but are not limited to, pMTNeo (Dickerson, I.M., et al. , unpublished) pCMVNeo (M.I. Rosenblatt and I.M. Dickerson, unpublished) and pcDNA3 (Invitrogen Corporation, San Diego, CA 92121) .
  • the present invention therefore also provides a host cell stably transformed or transfected with a vector containing a nucleic acid encoding CGRP-RCP.
  • the host cells may be prokaryotic cells such as E. coli, or eukaryotic such as animal, plant, insect and yeast cells.
  • the host cells are stably transformed or transfected by known procedures (see U.S. Patent Nos.4,704,362, 4,366,246, 4,425,437, 4,356,270, and 4,571,421).
  • amplification protocols may be used to obtain cells that produce high levels of recombinant proteins.
  • Other expression systems based on virus vectors may be used for extremely high level expression, but only transiently (e.g. baculovirus, Sindbis virus) .
  • the host cells may be screened for clones which produce the recombinant CGRP-RCP by known procedures such as Coomassie blue staining and Western blotting.
  • the recombinantly expressed protein may be obtained as a crude lysate or can be purified or partially purified by standard protein purification procedures known in the art such as differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and the like.
  • the CGRP-RCP has an amino acid sequence comprising the sequence shown in SEQ ID NO:4, or is a biologically active analogue thereof.
  • biologically active analogue thereof refers to proteins having substantially identical sequences to the sequence contained in SEQ ID NO:4, or to peptide fragments of such proteins, and which elicit CFTR activity in Xenopus oocytes as described in the Examples section.
  • proteins include, but are not limited, to any protein or polypeptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more amino acid residues have been conservatively substituted with a functionally similar residue.
  • conservative substitutions include, for example, the substitution of one non-polar (i.e. hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (i.e.
  • hydrophilic residue for another such as a substitution between arginine and lysine, between glutamine and asparagine, or between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • the isolated and purified CGRP- RCP of the present invention includes all members of the CGRP-RCP family.
  • modified forms of CGRP-RCP may be engineered and produced.
  • truncated or mutated forms of the protein can be produced by methods known to those of ordinary skill in the art to define functional domains of the CGRP-RCP and secreted forms can be engineered for the production of soluble CGRP-RCP.
  • the present invention also provides a method for detecting CGRP-RCP in a biological sample using antibodies which specifically react with CGRP-RCP.
  • antibodies includes monoclonal or polyclonal antibodies.
  • Exemplary antibody molecules for use in the detection methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobul ⁇ in molecules, or those portions of an immunoglobulin mole ⁇ cule that contain the antigen binding site, including those portions known in the art as F(ab) , F(ab') , F(ab')2, and F(v) .
  • antibodies specific to each different member of the CGRP-RCP family, or to peptide fragments thereof may be used to detect expression of their corresponding CGRP-RCP family member.
  • the complete protein, or peptide fragments thereof, of the particular members of the CGRP-RCP family may be utilized as an immunogen, with or without a carrier molecule, to produce these antibodies.
  • carrier molecules includes human albumin, bovine albumin, keyhole limpet hemo-cyanin, and the like.
  • Polyclonal and monoclonal antibodies, or their fragments may be produced by methods known in the art, or by genetic engineering (Kohler and Milstein (1975) Nature. 256: 495-497; Campbell “Monoclonal Antibody Technology, the “Production and Characterization of Rodent and Human Hybridomas” in Burdon, et al. (eds.) (1985) "Laboratory Techniques in Biochemistry and Molecular Biology, " Volume 13, Elsevier Science Publishers, Amsterdam).
  • the polyclonal or monoclonal antibodies may be formulated as pharmaceutical compositions for use m vivo or the antibodies may be used in immunoassays to detect the CGRP-RCP in biological samples.
  • the method of detecting CGRP-RCP comprises the steps of: (a) contacting the biological sample with an antibody; and (b) detecting the formation of a complex between the protein and the antibody.
  • the immunoassays are used to detect different members of the CGRP-RCP family in the biological sample using their respective antibodies.
  • Suitable immunoassays include, but are not limited to, immunoprecipitation, radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, and immunohistochemical assay.
  • Such assays may be direct, indirect, competitive or noncompetitive as described in the art. (In “Principles and Practice of Immunoassay” (1991) Christopher P. Price and David J. Neoman (eds), Stockton Press, New York, New York; Ausubel, et al. (eds) (1991) in "Current Protocols in Molecular Biology” John Wiley and Sons, New York, New York) .
  • the present invention also provides methods for identifying a ligand capable of binding to the CGRP receptor.
  • the method comprises (a) injecting Xenopus oocytes with a nucleic acid molecule encoding CGRP-RCP; (b) contacting the transformed oocytes with a candidate ligand; and (c) detecting the formation of a complex between CGRP receptor and the ligand. Detection of ligand binding may be measured by a variety of assays, including but not limited to, measurements of CFTRC1 " currents, measurements of CGRP binding, and cAMP immunoassays (to measure ligand-induced AMP response) .
  • the CGRP-RCP utilized in the ligand-screening assays is preferably recombinant CGRP-RCP.
  • ligand refers to any molecule that may interact with CGRP receptor.
  • the ligand may be naturally occurring protein, or peptide, or it may be a synthetically or recombinantly produced protein or peptide.
  • the ligand may also be a nonprotein molecule that acts as a ligand when it interacts with the CGRP receptor.
  • the ligands are preferably non-peptide ligands. Interactions between the ligand and CGRP receptor include, but are not limited to, covalent or noncovalent interactions.
  • the present invention also provides a method for screening for drugs capable of acting as agonists or antagonists to CGRP which comprises the steps of: (a) contacting cells which express the CGRP receptor with candidate drugs; and (b) evaluating the biological activity mediated by said contact.
  • the cells which express the CGRP receptor may be Xenopus oocytes injected with CGRP-RCP as the Examples presented herein demonstrate that injection of CGRP-RCP cRNA can confer a functional CGRP receptor to the oocyte.
  • drug includes but is not limited to proteins, peptides, agents purified from conditioned cell medium, organic molecules, inorganic molecules, antibodies, oligonucleotides, or analogs of the ligand described above.
  • the drug may be naturally occurring or synthetically or recombinantly produced.
  • biological activity means the triggering of the CGRP receptor protein to increase intracellular levels of cAMP.
  • this increase in cAMP levels may be evaluated by transfecting cells expressing CGRP receptor protein with a CFTR-containing plasmid, and measuring the triggering effect of specific drugs on the CGRP receptor protein by measuring CFTR-C1 " current in cells.
  • alterations in cAMP levels can be measured by cAMP radioimmunoassay.
  • the present invention therefore provides agonists or antagonists of CGRP identified using the methods above.
  • the present invention further provides for therapeutic and/or prophylactic methods which utilize the agonists and antagonists of the invention.
  • CGRP is one of the most potent vasodilators known, (Brain, S.D. et al (1985) Nature 313:54- 56) agonists of CGRP would be useful in dilating blood vessels and hence in lowering blood pressure.
  • the present invention relates to a method for treating hypertension in a subject comprising administering to the subject an amount of an agonist of CGRP effective to lower the blood pressure of the subject. It is believed that since different subtypes of CGRP receptor have been pharmacologically demonstrated on large versus small caliber blood vessels (Foulkes, R. et al (1991) Eur. J.
  • CGRP release from the trigeminal ganglia into the aqueous humor of the eye in response to inflammation coincides with an increase in intraocular pressure (Unger, W.G. et al (1985) J. Ocular Pharmacol.. 1:189-199); and Krootila, K. et al (1992) Curr. Eye Res.. 11:307-314) which is symptomatic of glaucoma and which is also a result of the inflammation that accompanies cataract surgery, it is contemplated that antagonists for CGRP would be useful in the treatment of inflammation/glaucoma in a subject.
  • the present invention therefore relates to a method for treating glaucoma in a subject comprising administering to the subject in need of such treatment a therapeutically effective amount of an antagonist to CGRP.
  • the present invention also relates to a method for treating premature labor in a subject comprising administering to a subject in need of such treatment, a therapeutically effective amount of an agonist of CGRP.
  • CGRP has been demonstrated to regulate the ability of immune cells in the skin (Langerhan cells) to present antigen, it is contemplated that agonists and antagonists of CGRP may be utilized in modulating immune responses.
  • the present invention also relates to a method for the treatment of pain comprising administering to a subject an amount of a CGRP antagonist effective to prevent the development of tolerance to morphine.
  • a CGRP antagonist effective to prevent the development of tolerance to morphine.
  • the route of administration will depend upon the intended use of the agonist or antagonist. For example, when agonists of CGRP are administered to a subject to treat hypertension, a preferred route of administration will be orally and, when antagonists of CGRP are administered to treat glaucoma, the antagonists will preferably be administered as eye drops.
  • Cesium-purified DNA was prepared from each pool, each pool was split into thirds and each third was restriction digested with a single enzyme: either Seal, Xmnl, or Nael; the three pools were then combined for transcription in vitro into capped cRNA using T7 RNA polymerase (Promega, Inc.) .
  • the CFTR- containing plasmid obtained from J. Riordan (Mayo Clinic, Scottsdale, Arizona; Rommens, J.M. et al. (1989) Science. 245:1059-1065; and Riordan, J.R. et al. (1989) Science.
  • Injected oocytes were incubated for 24-72 hrs at 19°C in OR2 media (Richter, J.D., & Smith, L.D. (1984) Nature, 309:378-380) to allow for protein synthesis and for transport of the receptor protein to the cell surface. Oocytes were then voltage clamped at -50 mV, and 10 '7 M rat ⁇ CGRP (Bachem California) was applied (rat c ⁇ CGRP was used for all experiments) . The pools were subdivided and retested until a candidate for the CGRP receptor was obtained.
  • Candidate cDNAs were sequenced using synthetic oligonucleotide primers with the dsCycle Sequencing kit (Gibco BRL, Bethesda, MD) .
  • Oocyte-CFTR dose response Oocytes were coinjected with 20 ng CGRP receptor cRNA and 20 ng CFTR cRNA, and after 24 hours individual oocytes were voltage clamped at -50 mV and exposed to increasing concentrations of CGRP ( IO *9 M through 10 '7 M) , with sequential applications to injected oocytes. All sequential applications were carried out at . >30 min intervals to avoid desensitization effects (Uezono, Y. et al. (1993) Receptor Channels. 1:233-241; and Rommens, J.M. et al. (1991) Proc. Natl. Acad. Sci. USA. 88:7500-7504) .
  • injected oocytes were separated into pools, and all oocytes of each pool were subjected to the same concentration of CGRP (10 "10 M through 10 "5 M) .
  • Antisense injections An antisense oligonucleotide containing a thiol-substituted backbone was synthesized to the 3' untranslated region of the CGRP-RCP cDNA (double underlined sequence, Fig 4a) . This antisense oligonucleotide was mixed just prior to injection with either CGRP-RCP cRNA or guinea pig cerebellar mRNA before injection. Oocytes were incubated for 48 hrs at 19°C and then tested with 10 '7 M CGRP.
  • Cerebellar mRNA or cRNA (diluted 1:100) was mixed with equal volumes of CFTR cRNA and either antisense oligonucleotide or water.
  • Northern blot Twenty ⁇ g of guinea pig cerebellar RNA was separated by denaturing agarose gel electrophoresis, transferred to a Nytran membrane (Schleicher & Schuell, Inc.) prehybridized for 4 hours at 42°C in 50% deionized formamide, 5X SSC, 4X Denhardt's solution, 0.1% SDS, 20 nm NaP0 4 pH6.5, and 100 ⁇ g/ml sonicated carrier DNA and then hybridized for 16 hours at 42°C with approximately IO 6 cpm/ml of 32 p-labeled probe ( 32 P-labeled random-primed probe made to the guinea pig cochlear CGRP receptor added to the prehybridization mix) .
  • Capped cRNA was transcribed in vitro from CGRP-RCP cDNA using the mMessage mMachine kit (Ambion, Inc.). One ⁇ g of cRNA was used for in vitro translation using rabbit reticulocyte extracts (Promega, Inc.), with or without canine pancreatic microsomes, (Promega Inc.) in the presence of 35 S-methionine (1000 Ci/mmol, Amersham, Inc.).
  • the sections were washed three times in PBS and incubated with ABC reagent (Vector Laboratories) , which binds streptavidin molecules conjugated to horseradish peroxidase (HRP) to each biotinylated site on the secondary antibody.
  • HRP horseradish peroxidase
  • This HRP was reacted with diaminobenzidine (DAB) to yield a brown reaction product and visualized using bright field microscopy.
  • DAB diaminobenzidine
  • EXAMPLE 1 Isolation of the CGRP-RCP Clone
  • an expression- cloning strategy was used that was based on an assay described by Uezono et al. ((1993) Receptors Channels. 1:233-241) .
  • This assay used cystic fibrosis transmembrane conductance regulator (CFTR) as a sensor for cAMP levels when expressed in Xenopus oocytes.
  • the CFTR is a protein kinase A (PKA) -activated chloride channel.
  • PKA protein kinase A
  • CFTR Cl current was demonstrated by application of forskolin to oocytes injected with CFTR complementary RNA (cRNA) .
  • cRNA CFTR complementary RNA
  • Forskolin raises intracellular cAMP levels, and as shown in Figure 2A, the addition of 20 ⁇ M forskolin resulted in the production of an inward current of approximately 1.0 microamperes ( ⁇ A) .
  • incubation with forskolin did not cause a membrane current (Uezono Y., et al . (1993) Receptors Channels. 1:233-241; Birnbaum, A.K., et al . (1994) Mol. Brain Res.. 28:72-80; Kartner, N., et al. (1991) Cell. 64:681-691).
  • the guinea pig organ of Corti library was screened in 20 pools of about 5000 colonies.
  • This effect was reversible, and could be repeated by reapplication of CGRP to the bath. No current could be elicited by CGRP in the absence of CFTR.
  • This positive pool was subdivided and retested, until, after six subdivisions, a candidate CGRP receptor was identified. During subsequent rounds of purification, single or multiple positive pools were identified.
  • CGRP-RCP Activation In Response To CGRP Oocytes were injected with CGRP-RCP cRNA and CFTR cRNA, incubated for 24 hrs, and sequentially exposed to increasing doses of CGRP. A representative set of current traces from a single oocyte is shown in Fig 3a to illustrate CGRP receptor activation in response to five doses of CGRP.
  • FIG. 3B shows data from a representative CGRP dose-response experiment. Oocytes were coinjected with CGRP-RCP cRNA and CFTR cRNA and subjected to eight doses of CGRP.
  • CFTR activation was specific to CGRP, as oocytes injected with CGRP-RCP cRNA would produce CFTR Cl " current upon application of 10 _9 M CGRP, but no detectable current was observed upon subsequent application of 10 "7 M calcitonin
  • VIP neuropeptide Y
  • Fig 3B ⁇ -endorphin
  • the ORF is preceded by a 48 bp 5' untranslated region (UTR) containing no upstream ATG codons, and is followed by a 1,232 bp 3' UTR containing stop codons distributed between all three reading frames.
  • the cochlear cDNA was confirmed by direct sequencing of a reverse transcription-polymerase chain reaction (RT-PCR) product amplified from cerebellar mRNA; cerebellum has been widely used as a source for CGRP binding studies ° (Stangle, D. , et al. (1991) . Biochem.. 30:8605-8611;
  • the 146 amino acid protein encoded by the ORF 5 ( Figure 4A) is largely hydrophilic ( Figure 4B) and has no homology to any reported receptor when searched against the GenBank database with BLAST software (Altschul, S.F., et al. (1990) J. Mol. Biol.. 215:403-410) .
  • RNA isolated from several tissues of guinea pig (cerebellum, cerebrum, heart, liver, lung, skeletal muscle and tongue) reported to have CGRP binding sites was analyzed by RNAase protection assays.
  • the CGRP-RCP was detected in all these tissues and the alternatively spliced subtype was detected in cerebellum only (data not shown) . This tissue distribution was confirmed with RT-PCR (data not shown) .
  • an antisense oligonucleotide was synthesized based on the sequence of the CGRP-RCP cDNA (primer 3, double underlined sequence in Fig 4a) .
  • the antisense oligonucleotide to the cochlear CGRP-RCP eliminated receptor activity from both the cochlear cRNA and cerebellar CGRP-RCP mRNA in the oocyte-CFTR assay, indicating that the cerebellum contains a CGRP receptor homologous to that of the cochlear form (Fig.7).
  • This conclusion agrees with the identical nucleotide sequence obtained from the cochlear cDNA and the cerebellar PCR product.
  • Connexin 38 is a membrane-bound gap-junction protein, which has been functionally removed from oocytes in antisense oligonucleotide experiments (Barrio, L.C, et al. (1991) Proc. Natl. Acad. Sci. USA. 88:8410-8414).
  • the connexin 38 antisense oligonucleotide did not alter CGRP receptor activity contained in the CGRP-RCP cRNA (data not shown) .
  • thiol-substituted oligonucleotides alone had no effect on CGRP-RCP expression in the oocyte.
  • calcitonin receptor-directed antisense oligonucleotides have been shown to have no effect on CGRP activity in oocytes injected with guinea pig brain mRNA (Sarkar, A. & Dickerson, I.M. (1994) Soc. Neurosci. Abstr.. 20:1346).
  • CGRP-RCP directed antisense oligonucleotides had no effect on calcitonin receptor activity from brain mRNA (Luebke, Dahl, Dickerson, unpublished observations) , indicating that CGRP-RCP and the calcitonin receptor do not interact.
  • the CGRP-RCP was shown to be present in cells innervated by CGRP-containing neurons.
  • CGRP-containing neurons Using an antibody against CGRP (MU33) , the efferent nerve fibers that terminate on outer hair cells of the basal two turns of the guinea pig cochlea were demonstrated to contain the ligand CGRP (Fig. 8, left panels) .
  • In situ hybridization with the cochlea CGRP-RCP antisense riboprobe showed that the mRNA for the CGRP-RCP is also only present in outer hair cells of the basal two turns of the guinea pig cochlea (Fig. 8, right panels) .
  • a cDNA from the cochlea of guinea pig that encodes a protein which confers responsiveness to CGRP in oocytes.
  • An antisense oligonucleotide made against the cochlear CGRP-RCP eliminates receptor activity induced in oocytes by cochlear receptor cRNA or by cerebellar mRNA.
  • In vitro translation yields a product consistent in size with the 146 amino acid receptor protein as predicted by the nucleotide sequence. In agreement with this size, a 17 kD protein that cross-links with CGRP has been identified from solubilized cerebellum (Stangle, D., Born, et al. (1991) . Biochem.. 30:8605-8611) .
  • CGRP-RCP cochlear CGRP-RCP
  • the cochlear CGRP-RCP is short and not obviously hydrophobic, it does not belong to the class of G-protein coupled receptors that contain seven membrane- spanning helices.
  • Two models could reconcile the primary structure of CGRP-RCP with reports that CGRP binding is coupled to G protein activation: First the CGRP-RCP could represent the complete CGRP receptor which would contain an atypical membrane-spanning domain, in which case it might resemble the mannose-6-phosphate receptor, which couples to G-proteins despite lacking the prototypical seven membrane-spanning helices (Okamoto, T. et al . (1990) Cell. 62:709-717) .
  • the CGRP-RCP could be part of a complex of proteins that constitute the CGRP receptor.
  • CNTF ciliary neurotrophic factor
  • IL-6 interleukin-6
  • Ip type I interferon
  • a small extracellular membrane-associated protein binds the ligand and interacts with a membrane- spanning protein for signal transduction, conferring specificity to a more generic signaling pathway.
  • the CGRP-RCP may be contributing specificity in a similar manner. In this scenario, binding of CGRP to its receptor may activate the CFTR in the oocyte assay via a membrane- associated kinase, either in conjunction with, or separetely from, the adenylate cyclase pathway depicted in Fig. 1.
  • the CGRP-RCP may couple the ligand-binding complex to the cellular signal transduction machinery. In the latter case, the CGRP-RCP is specific fo only CGRP, as no other tested ligand could activate the oocyte-CFTR assay when tested with the CGRP-RCP.
  • CGRP-RCP is part of a complex of proteins, it could be part of a complex with a known protein such as the RDC-l receptor (Kapas, S. et al . (1995) Biochem. Biophys. Res. Comm.. 217:532-538) or the CGRP1 receptor (Aiger, N. et al., J. Biol. Chem. (1996)) 271:11325-11329) or, with a novel unknown protein.
  • Example 8 The inhibitory effects of calcitonin gene related peptide (CGRP) on smooth muscle contraction, including uterine smooth muscle, have been well characterized (Samu ⁇ lson UE, et al. (1985) Neurosci. Iett. , 62:225; Shew RL, et al. (1990) Peptides. 11 :583-589; and Tritthart HA. et al. (1992). Annals N Y Acad Sci. 657:216-22723,24,28). Additionally, CGRP-containing nerve fibers are present in the human uterus and CGRP inhibits spontaneous and evoked contractions in the human uterus and fallopian tubes (Haase EB, et al.
  • CGRP-containing nerve fibers are present in the human uterus and CGRP inhibits spontaneous and evoked contractions in the human uterus and fallopian tubes (Haase EB, et al.
  • CGRP is therefore a candidate for the proposed inhibitory regulator of myometrial contractility during gestation.
  • Timed-pregnant female CD1 mice were obtained from Charles River Laboratories (Wilmington, MA) . Strips of myometrium were dissected from the longitudinal muscle layer of mice from various gestational stages and post partum (embryonic day 6 through post natal day 2: E6-PN2) and were placed in Krebs-bicarbonate solution (mM: NaCl, 119; NaHC0 3 , 25; MgS0 4 1.2; KCl, 3.6; KH 2 P0 4 , 1.2; CaCl 2 , 2.5; glucose, 11; pH 7.4) . Myometrial strips were mounted between forceps in a 0.5 ml perfusion chamber.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Tri Reagent Molecular Research Center, Inc.
  • Tissue samples were homogenized in Tri Reagent (100 mg tissue/ml reagent) using a Polytron homogenizer (Brinkman) .
  • Total RNA and protein were isolated according to the manufacturers instructions (Molecular Research Center Inc.).
  • Polyadenylated (Poly A+) RNA was selected from total RNA using the PolyATract mRNA Isolation System (Promega) .
  • Protein samples were resuspended in 0.1% SDS plus proteinase inhibitors (50 ⁇ g/ml Lima Bean Trypsin Inhibitor, 2 ⁇ g/ml Leupeptin, 16 ⁇ g/ml Benzamidine, 2 ⁇ g/ml Pepstatin A) and the protein content was determined using the Pierce Micro-BCA kit (Pierce) .
  • proteinase inhibitors 50 ⁇ g/ml Lima Bean Trypsin Inhibitor, 2 ⁇ g/ml Leupeptin, 16 ⁇ g/ml Benzamidine, 2 ⁇ g/ml Pepstatin A
  • RT-PCR Reverse Transcription-Polymerase Chain Reaction
  • Reverse transcription reactions primed with the degenerate primer DRES-1 (TCYTCNACCATNARYTGNATYTCNAC) were performed on 200 ng of mouse uterine mRNA.
  • the resulting first strand cDNA was purified and used as template for PCR using primers DRES-2 (C UACUACUAC UATG NARYTTYTCNGCYTTNGTNARYTTRTG) and D RES-3 (CAUCAUCAUCAUGTNTTYCARYTNYTNACNGAYYTNAA) .
  • the reaction was first cycled three times: 1 min at 94°C; 1 min at 37°C, a 2 min ramp to 72°C and then 1 min at 72°C; followed by 25 cycles of PCR using standard conditions: 1 min at 94°C, 1 min at 65°C, 1 min at 72°C.
  • Degenerate primers DRES-2 and DRES-3 were synthesized with 5' tails containing uracil residues (Gomez-Saladin E, et al. (1994) Cell Mol Neurobiol. 14:9-24), as described in the CloneAmp protocol (Gibco- BRL) .
  • the PCR amplimers were annealed into the plasmid pAmp-1, using the CloneAmp System (GIBCO-BRL) , and the sequence determined using the dsDNA Cycle Sequencing System (GIBCO-BRL) .
  • the sequence obtained from degenerate PCR amplimers was used to design primers specific for the mouse CGRP-RCP: MRES-5B (5' TCATTGCTGTGAGGAATTCTTGGA 3') and MRES-6B (5' GAGCAGCGGAAGGAGAGTGGGAAGAAC 3').
  • CGRP-RCP Cloning of CGRP-RCP from mouse uterus was performed using the Marathon Race cDNA kit (Clontech, Inc.) . Briefly, 1 ⁇ g mRNA from mouse uterus was used for reverse transcription, using a "lock-dock" oligo-dT primer (Clontech, Inc.). The resulting first-strand cDNA was then used as template for second strand cDNA synthesis, using standard conditions (Clontech, Inc.). The cDNA library was blunt-ended with T4 DNA polymerase, and Marathon adaptors were ligated to the double-stranded cDNA.
  • RACE was then carried out on the uterine cDNA library using primer MRES-5B and Marathon Adaptor Primer API (3' -RACE), or MRES-6B and API (5' -RACE).
  • the overlapping 5'and 3'- RACE products were ligated into the plasmid pCRII (Invitrogen) and the sequence was determined.
  • pCRII Invitrogen
  • the sequence information obtained from 5'- and 3' -RACE was used to design primers that flank the CGRP-RCP open reading frame (ORF) .
  • the upstream primer (T7-M1) incorporated the T7 promoter at its 5' end.
  • the primers had the following sequences: T7-M1:
  • Mouse uterine mRNA was reverse-transcribed and used for PCR with primers T7-M1 and MRES-17.
  • the resulting PCR product was transcribed in vi tro using the Ambion mMESSAGE mMACHINE kit, and the cRNA was injected into oocytes or was translated in vi tro in the presence of ["s] methionine using the Rabbit Reticulocyte Lysate
  • the translation product was resolved by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) , and then the gel was treated with Amplify (Amersham) , dried, and exposed to x-ray film.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • Uterine mRNA (2 ,ug) from embryonic day 6 through post natal day 2 was analyzed by electrophoresis on a denaturing 1% agarose/6% formaldehyde gel, transferred and UV cross-linked onto Nytran membranes (Schleicher & Schuell) .
  • Membranes were hybridized for 16 hrs with a 148-bp, - ⁇ P-labeled mouse CGRP-RCP probe and washed as described (luebkel996) .
  • Membranes were exposed to Kodak Biomax film (Eastman Kodak, Rochester, NY) with an intensifying screen at -80°C Membranes were subsequently stripped of CGRP-RCP probe and re-hybridized with a ⁇ 2 P-labeled glyceraldehyde phosphate dehydrogenase
  • GPDH Global Densitometer
  • Probes for CGRP-RCP were synthesized by PCR on mouse CGRP-RCP cDNA using primers MRES-5B and MRES-6B in the presence of [ 32 P] - ⁇ dCTP (3000 Ci/mmol, New England nuclear, Inc.), using the PCR Radioactive Labeling System
  • E6-PN2 Seventy five ⁇ g of protein isolated from the uteri of animals (E6-PN2) was resolved by 15% SDS-PAGE and electrotransferred to Immobilon-P membranes (Millipore) . Following the Rapid Immunodetection of Blotted Proteins protocol (Millipore Application Note, RP562) , membranes were air dried and then incubated for two hours with 1:500 dilution of CGRP-RCP polyclonal antibody R82, which was raised against the peptide sequence "TDLKDQRPRESGKMRHSAG" .
  • the membranes were next washed twice (10 sec) in 0.01 M Tris buffered saline plus 0.05% Tween-20 (TBS-T) and incubated with 1:10,000 dilution of goat anti-rabbit antibody conjugated to horseradish peroxidase (Amersham, Inc.) for 30 min, and washed twice with TBS-T (10 sec) .
  • TBS-T Tris buffered saline plus 0.05% Tween-20
  • the amount of CGRP-RCP in each lane was detected by chemiluminescence using the ECL Western Blotting Detection System (Amersham, Inc.) .
  • CGRPRCP mouse CGRP-receptor component protein
  • the uterine CGRP receptor was characterized utilizing an oocyte expression assay, in which the cystic fibrosis transmembrane conductance regulator (CFTR) was used as a sensor for intracellular cAMP levels (Uezono Y, et al. (1993). Receptors & Channels. 1 233-241). It was previously determined that mRNA isolated from tissue containing CGRP receptors (cerebellum) co-injected into oocytes with CFTR cRNA, conferred CGRP responsiveness (18) . The protein mediating this CGRP responsiveness has been identified in guinea pig as the CGRPreceptor component protein (CGRP-RCP) (Luebke AE, et al. (1996) Proc.
  • CGRP-RCP CGRPreceptor component protein
  • mouse CGRP-RCP homologue was isolated using degenerate RT-PCR.
  • Mouse uterine mRNA was reverse transcribed using the downstream degenerate primer DRES-1 and the resulting first strand cDNA was used as template for PCR using primers DRES-2 and DRES-3 (Fig. 14) .
  • the resulting PCR amplimer was cloned and sequenced, and used to design primers for 5'- and 3'- RACE to obtain the full length mouse CGRPRCP cDNA.
  • the 1.8 kb cDNA of the mouse CGRP-RCP contains a 444 bp ORF preceded by a Kozak translation initiation consensus sequence (Kozak M (1987) Nucleic Acids Res. 15, 8125-8148) (Fig. 14) .
  • the 148 amino acid protein encoded by the ORF is largely hydrophilic and highly conserved between mouse and guinea pig (Fig. 15) .
  • the in vi tro transcribed cRNA produced a 20 kDa protein when translated in vi tro (Fig. 16) , in agreement with the 148 amino acid ORF.
  • CGRP-RCP expression in occytes The oocyte-CFTR expression assay was used to verify CGRP-RCP function.
  • the CGRP-RCP cDNA was found to be genetically unstable, therefore, the cDNA was not transcribed directly. Instead, mouse uterine mRNA was reverse transcribed and amplified by PCR using primers T7-M1 and MRES-17 which flank the ORF (Fig. 2). This also verified that the short ORF was sufficient to confer function.
  • the resulting PCR product was usod as template for in vi tro transcription. Oocytes were co-injected with 50 ng CFTR cRNA and 50 ng CGRP-RCP cRNA.
  • Oocytes were voltage clamped at -50 mV and incubated with lOOnM CGRP.
  • Oocytes expressing the protein encoded by the CGRP-RCP ORF produced an inward current when incubated with CGRP (Fig. 5 a) .
  • the response was specific for CGRP, since other peptides tested such as calcitonin, amylin, and NPY produced no response (data not shown) .
  • Co-injection of an antisense oligonucleotide made to the cloned mouse CGRP-RCP (MRES-6B, Fig. 14) , eliminated receptor activity from oocytes injected with uterine mRNA (Fig. 17B) indicating that the CGRP-RCP in uterine mRNA mediated CGRP responsiveness.
  • CGRP-RCP mRNA was quantified by densitometric scanning of three northern blots and normalized to GAPDH mRNA level for each time point. Expression of CGRP-RCP mRNA did not vary significantly throughout gestation and post-partum (Fig. 18B) .
  • CGRP-RCP is a receptor molecule by itself or whether it associates with other cellular proteins to form a receptor complex that can be activated by CGRP.
  • RDCl and CGRPl transmembrane-spanning receptors
  • CGRP-RCP has been identified in cells innervated by CGRP-containing neurons in cochlea and cerebellum (Luebke AE, et al. (1996) Proc. Natl. Acad. Sci. (USA) 93:3455-3460, and Luebke AE, et al. (1996) Neurosci.
  • CGRP-RCP is an essential component of the CGRP signaling mechanism.
  • CGRP-RCP is an essential component of the CGRP signaling mechanism.
  • CGRP and its receptor-signaling complex probably play a key role in the control of myometrial contractility activity.

Abstract

L'invention concerne le clonage de la CGRP-RCP (protéine composante du récepteur du CGRP-peptide lié au gène de la calcitonine) et l'utilisation dudit clone pour mettre au point des dosages diagnostiques destinés à la CGRP-RCP, ainsi que pour cribler des agonistes et des antagonistes du CGRP.
PCT/US1997/006321 1996-04-15 1997-04-15 Clone moleculaire de proteine composante du recepteur du cgrp et son utilisation WO1997041223A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0821061A2 (fr) * 1996-07-23 1998-01-28 Smithkline Beecham Corporation Facteur composant du récepteur du peptide apparenté au gène de la calcitonine
US20110189205A1 (en) * 2008-07-09 2011-08-04 University Of Rochester Methods of treating cancer using an agent that modulates activity of the calcitonin-gene related peptide ("cgrp") receptor
US20150361173A1 (en) 2005-11-14 2015-12-17 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9328167B2 (en) 2008-03-04 2016-05-03 Labrys Biologics, Inc. Methods of treating chronic pain
US9896502B2 (en) 2014-03-21 2018-02-20 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
WO2018045083A1 (fr) * 2016-09-02 2018-03-08 Soares Christopher J Utilisation d'antagonistes du récepteur cgrp en neuroprotection et pour le traitement de troubles neurologiques
US10370425B2 (en) 2012-01-26 2019-08-06 Christopher Joseph Soares Peptide antagonists of the calcitonin CGRP family of peptide hormones and their use
US10392434B2 (en) 2016-09-23 2019-08-27 Teva Pharmaceuticals International Gmbh Treating refractory migraine
US10556945B2 (en) 2014-03-21 2020-02-11 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
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EP0821061A2 (fr) * 1996-07-23 1998-01-28 Smithkline Beecham Corporation Facteur composant du récepteur du peptide apparenté au gène de la calcitonine
EP0821061A3 (fr) * 1996-07-23 1999-05-26 Smithkline Beecham Corporation Facteur composant du récepteur du peptide apparenté au gène de la calcitonine
US9340614B2 (en) 2005-11-14 2016-05-17 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9890211B2 (en) 2005-11-14 2018-02-13 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide
US20150361173A1 (en) 2005-11-14 2015-12-17 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9266951B2 (en) 2005-11-14 2016-02-23 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9328168B2 (en) 2005-11-14 2016-05-03 Labrys Biologics, Inc. Methods of using anti-CGRP antagonist antibodies
US9890210B2 (en) 2005-11-14 2018-02-13 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide
US10329343B2 (en) 2005-11-14 2019-06-25 Teva Pharmaceuticals International Gmbh Methods for treating headache using antagonist antibodies directed against calcitonin gene-related peptide
US9346881B2 (en) 2005-11-14 2016-05-24 Labrys Biologics, Inc. Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US9365648B1 (en) 2005-11-14 2016-06-14 Labrys Biologics, Inc. Methods of using anti-CGRP antagonist antibodies
US9884908B2 (en) 2005-11-14 2018-02-06 Teva Pharmaceuticals International Gmbh Methods for treating headache using antagonist antibodies directed against calcitonin gene-related peptide
US9884907B2 (en) 2005-11-14 2018-02-06 Teva Pharmaceuticals International Gmbh Methods for treating headache using antagonist antibodies directed against calcitonin gene-related peptide
US9328167B2 (en) 2008-03-04 2016-05-03 Labrys Biologics, Inc. Methods of treating chronic pain
US10323085B2 (en) 2008-03-04 2019-06-18 Teva Pharmaceuticals International Gmbh Methods of treating fibromyalgia
US8394767B2 (en) * 2008-07-09 2013-03-12 University Of Rochester Methods of treating cancer using the calcitonin-gene related peptide (“CGRP”) receptor antagonist CGRP8-37
US20110189205A1 (en) * 2008-07-09 2011-08-04 University Of Rochester Methods of treating cancer using an agent that modulates activity of the calcitonin-gene related peptide ("cgrp") receptor
US10597448B2 (en) 2009-08-28 2020-03-24 Teva Pharmaceuticals International Gmbh Methods for treating visceral pain associated with interstitial cystitis by administering antagonist antibodies directed against calcitonin gene-related peptide
US10370425B2 (en) 2012-01-26 2019-08-06 Christopher Joseph Soares Peptide antagonists of the calcitonin CGRP family of peptide hormones and their use
US10556945B2 (en) 2014-03-21 2020-02-11 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US10519224B2 (en) 2014-03-21 2019-12-31 Teva Pharmaceuticals International Gmbh Treating headache comprising administering an antibody to calcitonin gene-related peptide
US9896502B2 (en) 2014-03-21 2018-02-20 Teva Pharmaceuticals International Gmbh Antagonist antibodies directed against calcitonin gene-related peptide and methods using same
US11555064B2 (en) 2014-03-21 2023-01-17 Teva Pharmaceuticals International Gmbh Treating headache comprising administering an antibody to calcitonin gene-related peptide
CN109922820A (zh) * 2016-09-02 2019-06-21 克里斯托弗·J·索尔斯 Cgrp受体拮抗剂在神经保护和神经系统疾病中的应用
JP2019530741A (ja) * 2016-09-02 2019-10-24 クリストファー ジェイ． ソアレス 神経保護及び神経疾患におけるｃｇｒｐ受容体アンタゴニストの使用
WO2018045083A1 (fr) * 2016-09-02 2018-03-08 Soares Christopher J Utilisation d'antagonistes du récepteur cgrp en neuroprotection et pour le traitement de troubles neurologiques
US11390654B2 (en) 2016-09-02 2022-07-19 Christopher Joseph Soares Use of CGRP receptor antagonists in neuroprotection and neurological disorders
KR102657418B1 (ko) * 2016-09-02 2024-04-15 크리스토퍼 제이 소레스 신경 보호 및 신경 질환에서의 cgrp 수용체 길항제의 용도
EP4316595A3 (fr) * 2016-09-02 2024-04-17 Christopher J. Soares Utilisation d'antagonistes du récepteur cgrp pour le traitement du glaucome
US10392434B2 (en) 2016-09-23 2019-08-27 Teva Pharmaceuticals International Gmbh Treating refractory migraine
US11028160B2 (en) 2016-09-23 2021-06-08 Teva Pharmaceuticals International Gmbh Treating refractory migraine
US11028161B2 (en) 2016-09-23 2021-06-08 Teva Pharmaceuticals International Gmbh Treating refractory migraine

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