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Molecular Cloning and Characterization of the Porcine Calcitonin Gene-Related Peptide Receptor

Departments of Molecular Genetics (N.A.E., J.E.A., J.M., D.J.B.), Gene Expression Sciences (A.M.S., S.G.), and Cardiovascular Pharmacology (J.D., N.A.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

Address all correspondence and requests for reprints to: Dr. Nabil A. Elshourbagy, Departments of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.

	Abstract

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Calcitonin gene-related peptide (CGRP) receptors (CGRP-Rs) are widely distributed throughout the central and peripheral nervous systems. A novel CGRP-R was identified from a porcine lung complementary DNA library. Sequence analysis indicated that the CGRP-R is 462 amino acids in length and shares 93% sequence identity with the human CGRP-R. Northern blot analysis indicated a messenger RNA species of 5.4 kilobases, which is abundantly expressed in the lung. Ligand binding studies of the cloned CGRP-R expressed in human embryonic kidney (HEK-293) cells showed the presence of high affinity receptor for CGRP with a K_d of 38.5 pM. The pharmacological profiles of various ligands competing for [¹²⁵I]CGRP binding to the expressed receptor were in accordance with those for the natural receptor. Binding of [¹²⁵I]CGRP to the expressed receptor was decreased in the presence of a nonhydrolyzable analog of GTP, guanosine 5' (-thio)-triphosphate. In functional studies, CGRP stimulated the activation of adenylyl cyclase with an EC₅₀ of 2.5 nM. The linear analog of CGRP, diacetoamidomethyl cysteine CGRP, did not affect adenylyl cyclase activity on its own or in the presence of CGRP. Furthermore, the CGRP receptor antagonists, CGRP-(8–37), inhibited the CGRP-mediated response in a competitive manner. Collectively, the binding and functional data demonstrate that we have cloned a porcine CGRP type 1 receptor. The availability of the CGRP-R complementary DNA will allow us to examine its participation in pathophysiological processes.

	Introduction

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CALCITONIN gene-related peptide (CGRP) is a naturally occurring, 37-amino acid peptide that is generated by tissue-specific alternate processing of calcitonin messenger RNA (mRNA) (1) and is widely distributed in the central and peripheral nervous systems (2). CGRP is localized predominantly in sensory afferent and central neurons and exhibits several biological actions, including vasodilation. CGRP is expressed in - and ß-forms that vary by one and three amino acids in the rat and human, respectively. CGRP and CGRPß display similar biological properties. When released from the cell, CGRP initiates its biological responses by binding to specific cell surface receptors that are predominantly coupled to the activation of adenylyl cyclase (3, 4). CGRP receptors (CGRP-Rs) have been identified and pharmacologically evaluated in several tissues and cells, including those of brain, cardiovascular, endothelial, and smooth muscle origin (2). Based on pharmacological properties, these receptors are divided into at least two subtypes, denoted CGRP₁ and CGRP₂ [according to the classification of Dennis and Quirion (5–7)]. Human (h) CGRP-(8–37), a fragment of CGRP that lacks seven N-terminal amino acid residues, is a selective antagonist of CGRP₁-Rs, whereas the linear analog of CGRP, diacetoamidomethyl cysteine CGRP ([Cys(ACM)2,7]CGRP), is a selective agonist of CGRP₂-Rs (6). We recently reported the cloning and characterization of complementary DNA (cDNA) encoding hCGRP-R type 1 (8, 9). Stable expression of this cDNA in human embryonic kidney (HEK-293) cells produced specific, high affinity binding sites for CGRP that displayed pharmacological and functional properties very similar to those of native CGRP₁-R. Exposure of these cells to CGRP resulted in an increase in cAMP production, which was inhibited in a competitive manner by CGRP₁-R antagonist hCGRP-(8–37).

We report here the cloning of a cDNA encoding the porcine CGRP-R that shares 93% protein sequence identity with the hCGRP-R. Binding and functional studies confirm that the cloned receptor is the porcine ortholog of hCGRP₁-R.

	Materials and Methods

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Materials
(2-[¹²⁵I]Iodohistidyl¹⁰)hCGRP (SA, 2000 Ci/mmol) was obtained from Amersham (Chicago, IL). hCGRP, hCGRP-(8–37), human adrenomedullin (ADM), salmon calcitonin, human calcitonin, endothelin, and porcine vasoactive intestinal peptide were purchased from Bachem Bioscience, Inc. (King of Prussia, PA). hCGRP-(9–37), hCGRP-(10–37), and hCGRP-(11–37) were synthesized at SmithKline Beecham Pharmaceuticals (King of Prussia. PA). The BCA (bicinchoninic acid) protein assay kit was obtained from Pierce Chemical Co. (Rockford, IL). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

Construction and screening of the cDNA libraries
A porcine lung cDNA library constructed in a ZAP express vector (10) was screened by hybridization using a ³²P-labeled human CGRP cDNA-coding sequence as a probe in 20% formamide, 5 x SSC (SSC is 150 mM NaCl and 15 mM sodium citrate), 5 x Denhardt’s solution, 0.1% SDS, and 0.2 mg/ml Escherichia coli transfer RNA at 42 C (11). Filters were finally washed with 2 x SSC-0.1% SDS at 42 C. Several positive recombinant clones were isolated and characterized. Preliminary sequence analysis showed that six of these clones encoded the porcine CGRP-R.

Nucleotide sequence analysis
Both strands of the cDNA insert encoding the porcine CGRP-R were sequenced using a modification of the dideoxy chain termination method (12) using the Sequenase II kit (U.S. Biochemical Corp., Cleveland, OH). The WI Genetics Computer Group Software package (13) was used to assemble composite sequences from the various fragments and for sequence analysis.

RNA blot analysis
For Northern analysis, polyadenylated RNA was isolated from various porcine tissues using the guanidinium thiocyanate acid-phenol method (14). One microgram of each RNA was fractionated on 1% agarose-formaldehyde gels (15) and transferred to nitrocellulose membranes. Hybridizations were performed at 42 C in 50% formamide, 5 x SSPE (0.75 M NaCl, 0.05 M NaH₂ PO₄, 0.06 M EDTA), 5 x Denhardt’s reagent (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA), 0.1% SDS, and 100 µg/ml yeast transfer RNA (16) using ³²P-labeled porcine CGRP receptor cDNA insert. The membranes were finally washed with 0.1 x SSPE-0.1% SDS at 50 C and exposed to x-ray film for 4 days at -70 C.

Stable expression of the porcine CGRP-R
A fragment containing the entire porcine CGRP-R cDNA (coding sequences only) was subcloned into the mammalian expression vector pCDN (17). Human embryonic kidney cells (HEK-293) were plated at 3 x 10⁵ cells/well in a six-well tissue culture plate in Eagle’s MEM supplemented with 10% FBS. The next day, cells were transfected in serum-free Eagle’s MEM with 2 µg plasmid DNA encoding the porcine CGRP-R gene using 5 µl Lipofectamine (Life Technologies, Gaithersburg, MD) following the manufacturer’s protocol. After 48 h, cells were diluted, transferred to 100-mm dishes, and selected in 0.4 mg/ml geneticin (Life Technologies) until visible foci formed (15 days). Colonies were picked, expanded, and tested for the expression of porcine CGRP-R by cAMP production. One of the six stable clones was characterized in detail. To confirm that the transfected HEK-293 clonal cell line expressed the porcine CGRP-R, oligonucleotide primers were synthesized corresponding to the amino-terminal (ATG GAG AAA AAG TAT ATC CTG TAT TTT C) and carboxyl-terminal (TTC CAT TTA AGT GTT CGC TTG GAT AG), and RT-PCR was used to clone the porcine CGRP-R cDNA from the HEK-293/CGRP-R transfected cell mRNA. As determined by DNA sequence analysis, the porcine CGRP-R was indeed expressed in transfected, but not vector-transfected, cells.

Membrane preparation
Cells were detached from culture flasks (T-150, Falcon) with 1 mM EDTA in Ca²⁺/Mg²⁺-free Dulbecco’s PBS, washed by centrifugation at 300 x g, and stored as frozen pellet. Crude membranes were prepared as we described previously for porcine lung membranes (18). In brief, the cells were suspended in ice-cold buffer containing 10 mM Tris-HCl (pH 7.4), 5 mM Na-EDTA, 0.1 mM phenylmethylsulfonylfluoride, 1.0 mg/ml bacitracin, and 0.1 mg/ml aprotinin (buffer A) and homogenized using a Dounce homogenizer (Kontes Co., Vineland, NJ). The homogenates were centrifuged at 47,000 x g for 20 min at 4 C; the membrane pellets were washed twice by centrifugation in buffer containing 20 mM Na-HEPES (pH 7.4), 5 mM MgCl₂, 2 mM Na-EGTA, and 0.1 mg/ml bacitracin (buffer B); and the membranes were resuspended in the same buffer at 5 mg/ml and stored frozen at -70 C. The protein concentration was measured by the Pierce BCA method using BSA as a standard.

Radioligand binding assays
Radioligand binding assays were performed as described previously (19). In saturation binding studies, increasing concentrations of [¹²⁵I]hCGRP were added and incubated in a total volume of 500 µl (50–100 µg membrane proteins/ml) for 30 min at 25 C. Nonspecific binding was determined in the presence of 1 µM unlabeled hCGRP. In competition binding studies, the membranes (50–100 µg membrane protein/ml) were incubated with increasing concentrations (1 pM to 1 µM) of competing ligand and 125–150 pM [¹²⁵I]hCGRP for 30 min at 25 C. The incubations were terminated by the addition of 2 ml cold wash buffer (0.9% NaCl) followed by rapid filtration over Skatron filtermates presoaked in 0.2% polyethyleneimine using a Skatron cell harvester (Skatron Instruments, Norway). All binding assays were performed in duplicate, and each experiment was repeated three or four times. Analysis of all binding data (determination of K_d, binding capacity, and K_i values) was performed by computer-assisted nonlinear least square fitting using GraphPad PRIZM (GraphPad Software, San Diego, CA).

Adenylyl cyclase activity
Adenylyl cyclase activity was measured in triplicate as the rate of conversion of [-³²P]ATP to [³²P]cAMP, as previously described (19). Membranes (40–60 µg protein) were incubated in triplicate in buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 1.2 mM ATP, 1.0 µCi [-³²P]ATP, 0.1 mM cAMP, 2.8 mM phosphoenolpyruvate, and 5.2 µg/ml myokinase in a final volume of 100 µl for 20 min at 30 C. The reactions were stopped with 1 ml of a solution containing cAMP (0.28 mM), ATP (0.33 mM), and 22,000 dpm [³H]cAMP. [³²P]cAMP was separated using sequential chromatography (Dowex and alumina columns) (20). Adenylyl cyclase activities were determined in the absence (basal) or presence of various concentrations of hCGRP (1 pM to 1 µM). The additive effect of the peptides on adenylyl cyclase was also determined. Forskolin (10 µM) was used as a positive control for the activation of adenylyl cyclase. The effects of various concentrations of hCGRP-(8–37), a CGRP-R antagonist, on hCGRP-mediated activation of adenylyl cyclase were also determined. Intracellular CGRP-mediated cAMP determinations were performed after seeding the stably transfected cells into six-well plates, as described previously (8).

	Results and Discussion

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Cloning of a cDNA encoding the porcine CGRP-R
The hCGRP-R cDNA, previously cloned in our laboratory (8), was used to probe a porcine lung cDNA library. Nucleotide sequence analysis of one of the hybridization positive clones (see Materials and Methods) revealed an open reading frame beginning at the methionine codon ATG (nucleotide position 1) and ending at a stop codon TGA (nucleotide 1382). Two potential in-frame ATG codons precede the open reading frame of the protein. However, the second ATG code most closely approximates a Kozak consensus translation initiation site (21); it is probably the translation initiation codon. Accordingly, the deduced polypeptide consists of 462 amino acid residues with a calculated molecular mass of approximately 50.8 kDa (Fig 1). This is similar to the size of the hCGRP-R, which is 461 amino acids in length with a calculated molecular mass of 50.7 kDa (Fig. 1). The single amino acid difference in length between the two species is an insertion of a glutamic acid residue (E; amino acid position 25) near the N-terminal region of the porcine CGRP-R. The hydropathy profile of the CGRP-R determined by the method of Kyte and Doolittle (22) indicates the presence of seven hydrophobic regions (16–28 amino acid residues in length), which are likely to be the membrane-spanning domains that form the seven-transmembrane motif found among various G protein-coupled receptors.

View larger version (67K): [in this window] [in a new window]   Figure 1. Alignment of the amino acid sequences of porcine and human CGRP-R cDNA clones. cDNA inserts encoding the porcine CGRP-R were sequenced using the dideoxy method (12). Deduced amino acid residues are indicated, beginning with the initiator methionine. The optional alignment of the deduced amino acid sequences of the porcine and human (8) clones were made with the WI GCG Software package. The regions identifying the positive transmembrane as domains I–VII are underlined and numbered sequentially.

mRNA size and tissue distribution
Northern blot hybridization analysis using the full-length cDNA as a hybridization probe revealed a mRNA species approximately 5.4 kilobases in size exclusively in the lung (Fig. 2). The porcine CGRP-R mRNA was not detected in the heart or cerebellum, which is consistent with the expression pattern observed for hCGRP-R (8).

View larger version (19K): [in this window] [in a new window]   Figure 2. Size and tissue distribution of CGRP-R mRNA in various porcine tissues. Polyadenylated RNA was prepared from the indicated tissues and then fractionated on 1% agarose-formaldehyde gel, blotted into nitrocellulose, and hybridized with 32P-labeled porcine CGRP-R cDNA. Human ß-actin was used as an internal standard for the amount of RNA loaded (data not shown). The position of a 5.4-kilobase band corresponding to the CGRP-R mRNA is indicated.

View larger version (15K): [in this window] [in a new window]   Figure 3. Saturation isotherms for binding of [125I]hCGRP to membranes prepared from HEK-293 cells expressing the recombinant porcine CGRP-R. Increasing concentrations of [125I]CGRP (3–175 pM) were incubated with the membranes (80 µg protein/ml) for 30 min at 25 C. Nonspecific binding was defined with 1 µM hCGRP. Specific binding was obtained by subtracting nonspecific binding from total binding. Inset, Scatchard transformation of specific binding.

View larger version (16K): [in this window] [in a new window]   Figure 4. Competition curves for representative CGRP analogs against [125I]hCGRP binding to HEK-293 cell membranes expressing the recombinant CGRP-R. A, hCGRP (), hCGRP-(8–37) (), hCGRP-(9–37) (), hCGRP-(10–37) (•), and hCGRP-(11–37) (). B, hCGRP (), hCGRPß (), hCGRP-(8–37) (), ADM (), Cys[ACM 2,7]CGRP (), salmon CT (*), angiotensin II (), and endothelin I (+). Increasing concentrations of the indicated ligands were added to membranes and incubated with [125I]hCGRP (50 pM) for 30 min at 25 C. Bound and free ligands were separated as explained in Materials and Methods.

View this table: [in this window] [in a new window]   Table 1. Ki (nanomolar concentrations) values for displacement of [125I]hCGRP binding

To assess CGRP-R-G protein interactions in HEK-293 cells expressing the porcine CGRP-R, competition binding experiments were performed with [¹²⁵I]CGRP-(8–37) and unlabeled CGRP in the presence and absence of a nonhydrolyzable GTP analog, guanosine 5' (-thio)-triphosphate (GTPS). As shown in Fig. 5, GTPS shifted the CGRP competition curve to the right, resulting in an increase in IC₅₀ value from 0.48 to 3.5 nM (n = 2). Analysis of the data showed that competition data by CGRP in both the absence and presence of GTPS were best fit by a model described by one class of binding sites with K_i values of 0.21 and 1.5 nM, respectively. These results show that binding of agonists to recombinant porcine CGRP-R was regulated by guanine nucleotides, presumably by an interaction with a guanine nucleotide-binding proteins.

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of GTPS on CGRP binding to the membranes from HEK-293 cells expressing recombinant porcine CGRP-R [125I]hCGRP-(8–37) was displaced by increasing concentrations of unlabeled hCGRP in the absence () or presence () of 100 µM GTPS. Data points are the average of three separate experiments.

View larger version (13K): [in this window] [in a new window]   Figure 6. Concentration-response curve of hCGRP on intracellular cAMP formation in HEK293 cells expressing the recombinant porcine CGRP-R. Data represent the mean ± SEM of two independent experiments performed in triplicate.

View larger version (17K): [in this window] [in a new window]   Figure 7. Stimulation of adenylyl cyclase activity in the membranes from HEK-293 cells expressing recombinant porcine CGRP-R by hCGRP and hCGRPß. The membranes (40–60 µg protein) were incubated in the presence of 10 µM GTP and increasing concentrations of hCGRP () and hCGRPß () at 30 C for 20 min and processed as explained in Materials and Methods.

View larger version (22K): [in this window] [in a new window]   Figure 8. Effect of hCGRP-(8–37) on hCGRP-mediated adenylyl cyclase activity. Increasing concentrations of hCGRP were added to the membranes from HEK-293 cells expressing recombinant porcine CGRP-R in the absence () or presence of the indicated concentrations of CGRP-(8–37), incubated in the presence of 10 µM GTP for 20 min at 30 C, and processed as explained in Materials and Methods.

	Acknowledgments

 
The authors are grateful to Dr. Ganesh Sathe for oligonucleotide synthesis, Stephanie Van Horn for sequence analysis, John Martin for synthesis of CGRP fragments, and Dr. Ponnal Nambi for critical review of the manuscript.

	Footnotes

 
¹ Present address: MedImmune, Inc., 35 West Watkins Mill Road, Gaithersburg, Maryland 20878.

Received May 20, 1997.

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