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Research ArticleCARDIOVASCULAR

Functional Calcitonin Gene-Related Peptide Subtype 2 Receptors in Porcine Coronary Arteries Are Identified as Calcitonin Gene-Related Peptide Subtype 1 Receptors by Radioligand Binding and Reverse Transcription-Polymerase Chain Reaction

Boyd R. Rorabaugh, Margaret A. Scofield, D. David Smith, William B. Jeffries and Peter W. Abel
Journal of Pharmacology and Experimental Therapeutics December 2001, 299 (3) 1086-1094;

Abstract

Calcitonin gene-related peptide (CGRP) receptors are classified into CGRP subtype 1 (CGRP1) and CGRP subtype 2 (CGRP2) based on the affinity of the antagonist, human α (hα)-CGRP8–37. hα-CGRP8–37 antagonizes CGRP1 receptor-mediated responses with high affinity (KB < 100 nM) and antagonizes CGRP2 receptor-mediated responses with low affinity (KB > 1 μM). CGRP2receptors have been previously reported to mediate relaxation of large porcine coronary arteries because this action is antagonized with low affinity by hα-CGRP8–37. In the present study, we used reverse transcription-polymerase chain reaction, radioligand binding, and values from our previously reported isolated tissue experiments to compare the CGRP receptor in porcine coronary arteries with the porcine CGRP1 receptor stably expressed in human embryonic kidney (HEK) 293 cells. We identified calcitonin receptor-like receptor and receptor activity modifying protein 1 mRNA in coronary arteries. We also found that the ligand binding characteristics of the CGRP receptor in coronary arteries and the cloned CGRP1 receptor were highly similar. KI values for hα-CGRP8–37 were 6.6 and 5.7 nM in porcine coronary arteries and the cloned CGRP1 receptor, respectively. The affinities (KB) of hα-CGRP8–37 and five other antagonists were 22- to 707-fold lower in functional experiments measuring relaxation of coronary arteries than in radioligand binding experiments. Despite this difference in absolute affinity values, there was a high correlation of the rank order of affinity for the antagonists determined by the two methods. Thus hα-CGRP8–37 antagonizes CGRP-induced relaxation of porcine coronary arteries with low affinity at the CGRP1 receptor. Taken together, these data do not support the existence of the CGRP2 receptor.

α-Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide generated by alternative splicing of the calcitonin gene primary transcript (Amara et al., 1982). β-CGRP, a second form of this peptide, is the product of a separate gene and varies from α-CGRP by three amino acids (Steenbergh et al., 1985). CGRP is widely distributed in central and peripheral regions of the nervous system and is involved in nociception (Yu et al., 1998), appetite suppression (Tannenbaum and Goltzmann, 1985), and regulation of gastrointestinal motility (Raybould, 1992). CGRP has also been proposed to play a role in inflammatory responses (Smith et al., 1993; Kilo et al., 1997), wound healing (Engin, 1998), and the maintenance of vascular tone (Gangula et al., 2000). Therapeutic uses for CGRP and its analogs are currently under investigation for the treatment of migraine headache (Doods et al., 2000) and other disorders.

CGRP produces its effects by activating specific G-protein-coupled receptors at the cell surface. Based on isolated tissue studies with the antagonist hα-CGRP8–37, two CGRP receptor subtypes, CGRP1 and the putative CGRP2 receptor, have been proposed to mediate the effects of CGRP. hα-CGRP8–37 reportedly antagonizes CGRP1 receptor-mediated responses with high affinity (KB < 100 nM) and antagonizes CGRP2 receptor-mediated responses with low affinity (KB > 1 μM) (Dennis et al., 1990; Mimeault et al., 1991; Wisskirchen et al., 1998). Agonists have also been used to classify CGRP receptors. It has been reported that [Cys(ACM2,7)]hα-CGRP is an agonist at the CGRP2 receptor and is inactive at the CGRP1 receptor (Dennis et al., 1989;Wisskirchen et al., 1998). These data are consistent with the existence of two CGRP receptor subtypes.

The CGRP1 receptor is formed by the coexpression of the calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP 1) (McLatchie et al., 1998). In nearly all tissues and cells that contain CGRP1 receptors, this receptor is coupled to an increase in intracellular 3′,5′ cyclic adenosine monophosphate. Activation of the CGRP1receptor is also reported to cause intracellular calcium mobilization (Aiyar et al., 1999) and the activation of extracellular regulated kinase, P-38 mitogen-activated protein kinase, and Jun kinase in some systems (Disa et al., 2000; Parameswaran et al., 2000). These signal transduction pathways have been studied in CGRP1receptor-transfected HEK 293 cells and in cells that endogenously express the CGRP1 receptor (Aiyar et al., 1999;Disa et al., 2000; Parameswaran et al., 2000; Rorabaugh et al., 2001).

In contrast to the CGRP1 receptor, the putative CGRP2 receptor has not been cloned or well-characterized. The CGRP2 receptor has only been identified by its low affinity (KB > 1 μM) for hα-CGRP8–37 in functional studies in which a response to CGRP is measured (Dennis et al., 1990). Foulkes et al. (1991) and Waugh et al. (1999) reported that CGRP-induced dilation of porcine coronary arteries is blocked with low affinity (KB = 5 μM) by hα-CGRP8–37. In addition, the putative CGRP2 receptor-selective agonist [Cys(ACM)2,7]hα-CGRP induces dilation of these arteries (Waugh et al., 1999). These studies establish that porcine coronary arteries have the prototypical characteristics of a tissue containing the putative CGRP2 receptor.

In the present investigation, we used RT-PCR, radioligand binding, and data from previously reported isolated tissue experiments to compare the CGRP2 receptor that is expressed in porcine coronary arteries with the porcine CGRP1 receptor that has been previously cloned and expressed in HEK 293 cells. Ligand affinities in isolated tissue experiments (KB) and in radioligand binding experiments (KI) were also compared. We found that the CGRP2 receptors that have been previously reported to mediate relaxation of isolated porcine coronary arteries are identified as CGRP1 receptors by radioligand binding and RT-PCR. These data do not support the proposal that there are two CGRP receptor subtypes.

Materials and Methods

Chemicals and Reagents.

Taq DNA polymerase, 10 times PCR buffer (200 mM Tris-HCl, pH 8.4, and 500 mM KCl), amplification grade DNase I, 100-base pair DNA ladder, TRIzol, minimum essential media, fetal bovine serum, and antibiotic/antimycotic (containing 10,000 units/ml penicillin G, 10,000 μg/ml streptomycin sulfate, and 25 μg/ml amphotericin B) were purchased from Invitrogen (Carlsbad, CA). Moloney murine leukemia virus reverse transcriptase was purchased from PerkinElmer (Foster City, CA). The pCRII cloning vector was purchased from Invitrogen, and [125I]hα-CGRP was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Na2Ca-ethylenediaminetetracetic acid, Tris(hydroxymethyl)aminomethane, Sigmacote, and other chemicals were obtained from Sigma (St. Louis, MO). HEK 293 cells stably expressing the porcine CGRP1 receptor were a generous gift from Dr. Allan R. Shatzman of SmithKline Beecham Pharmaceuticals (King of Prussia, PA).

Peptide Synthesis.

Adrenomedullin, calcitonin, hα-CGRP8–37, hα-CGRP, and [Cys(ACM)2,7]hα-CGRP were purchased from Peninsula Laboratories (San Carlos, CA). All other peptides were synthesized by solid phase methods and purified by reversed phase high-performance liquid chromatography as previously described (Li et al., 1997; Smith and Hanly, 1997; Saha et al., 1998; Rist et al., 1999). The structure of these peptides was verified by amino acid analysis and electrospray ionization-mass spectrometry.

RNA Isolation.

A fresh porcine heart was obtained from a local slaughterhouse and transported to the laboratory in ice-cold phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH = 7.3). The left circumflex coronary artery was isolated and cleaned of fat and connective tissue with the aide of a dissecting microscope, and the endothelium was removed by gentle scraping with a number 22 scalpel blade. The artery was wrapped in aluminum foil, frozen at −70°C, and pulverized with a hammer. TRIzol reagent was used to isolate total RNA from approximately 100 mg of pulverized artery according to the manufacturer's protocol. The RNA was dissolved in 20 μl of RNase-free water containing DNase I buffer and 5 units of amplification grade DNase I. The DNase I was removed by adding an equal volume of TRIzol and repeating the RNA isolation procedure. The RNA was dissolved in RNase-free water and stored at −70°C.

RT-PCR.

RT-PCR was used to identify CRLR mRNA in porcine coronary arteries with the primers shown in Fig.1. These primers were designed based on the porcine CRLR complementary DNA (cDNA) sequence provided by Dr. Allan R. Shatzman of SmithKline Beecham Pharmaceuticals. CRLR mRNA was initially detected in porcine coronary artery using primers 2 and 4 (Fig. 1). cDNA was synthesized by reverse transcription in a 10-μl reaction volume containing 1 times PCR buffer, 3 μg of RNA, 5 mM MgCl2, 1 mM dNTP mixture, 25 pmol of antisense primer, and 25 units of Moloney murine leukemia virus reverse transcriptase. The reaction was incubated in a PerkinElmer 2400 thermocycler at 42°C for 50 min followed by a 5-min incubation at 99°C. PCR was conducted in a 100-μl reaction volume containing 1 times PCR buffer, 3 mM MgCl2, 0.2 mM dNTP mixture, 50 pmol of sense primer, 50 pmol of antisense primer, 10 μl of cDNA, and 2.5 units of Taq DNA polymerase. PCR conditions included an initial cDNA denaturation step at 95°C for 5 min followed by 30 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 30 s) of PCR and a final extension period of 7 min at 72°C. Primers 2 and 5 (Fig. 1) were used to amplify the 3′ end of the CRLR mRNA coding region using the PCR conditions described above. Primers 1 and 3 (Fig. 1) were used to amplify the 5′ end of the CRLR mRNA coding region. PCR conditions for this pair of primers were the same as those described above except that the annealing temperature was changed to 50°C.

Figure 1
Figure 1

Primer sequences and their relative positions on the coding region of the CRLR mRNA. The solid line represents the porcine CRLR mRNA coding region and the horizontal open bar represents the position of the 223-base pair RT-PCR product shown in Fig. 2A. Sense primers are designated by vertical open boxes, and antisense primers are designated by vertical solid boxes. Roman numerals indicate regions of the mRNA that code for the transmembrane regions of the CRLR. A, adenosine; T, thymine; G, guanine; C, cytosine; R, adenosine/guanine degeneracy; and Y, thymine/cytosine degeneracy.

Sense (5′-GAC CAT CAG GAG CTA TAA AGA CC-3′) and antisense (5′-TGC CAG ACC ACC AGT GCG GTC-3′) primers were designed based upon the porcine RAMP 1 cDNA sequence (GenBank accession number AF312385). These primers were used to detect RAMP 1 mRNA in coronary arteries using the method described above. However, the annealing temperature was adjusted to 54°C, and 40 cycles of PCR were used. The products of all RT-PCR reactions were visualized on ethidium bromide-stained 1.5% agarose gels and subcloned into the pCRII vector. Both DNA strands of each RT-PCR product were sequenced using an Applied Biosystems 373 DNA sequencer (Foster City, CA). DNA sequences were analyzed using the Wisconsin Package version 10.1 (Genetics Computer Group, Madison, WI) software.

Cell Culture.

HEK 293 cells stably expressing the porcine CGRP1 receptor were grown in T-175 culture flasks in minimum essential medium that was supplemented with fetal bovine serum (10%), penicillin G (100 units/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml). The flasks were placed in a humidified incubator in an atmosphere of 5% CO2/95% air and maintained at 37°C. The cells were grown to confluence and then harvested for membrane preparations as described below.

Membrane Preparations.

Culture media was removed from confluent cells, and the cells were rinsed three times with 25 ml of ice-cold phosphate-buffered saline. Cells were dislodged from the flask with a cell scraper in the presence of 10 ml of ice-cold phosphate-buffered saline and centrifuged at 4°C for 5 min at 1000g. The pellet was suspended in 25 ml of buffer A (50 mM Tris-HCl and 5 mM Na2Ca-ethylenediaminetetracetic acid, pH 7.4) by vortexing and then homogenized with a glass-Teflon homogenizer. The homogenate was centrifuged at 100,000g for 30 min in a Beckman L5–50 ultracentrifuge. The pellet was washed twice by homogenization in 25 ml buffer B (50 mM Tris-HCl, 100 mM NaCl, and 5 mM MgCl2, pH 7.4), followed by centrifugation at 4°C for 30 min at 100,000g. The supernatant was removed, and the dry pellet was stored for up to 1 month at −70°C. Protein content of the final pellet was determined by the method of Lowry et al. (1951).

Fresh pig hearts were obtained from a local slaughter house and transported to the laboratory in ice-cold phosphate-buffered saline. The left circumflex, right circumflex, and anterior descending coronary arteries were removed and cleaned of fat and connective tissue with the aid of a dissecting microscope. The outside diameter of all coronary arteries used in this investigation was >1 mm. The arteries were cut open, and the endothelium was removed by gentle scraping with a number 22 scalpel blade. The arteries were cut into small pieces with scissors and homogenized in 25 ml of buffer A with an Ultra-Turrax T25 tissue homogenizer for 3 min at 24,000 rpm. The homogenate was centrifuged at 4°C for 10 min at 1000g to remove particulate debris. The supernatant was centrifuged at 4°C for 30 min at 100,000g, and the resulting pellet was washed twice in buffer B as described above for membranes from HEK 293 cells. Protein content of the final pellet was determined by the method of Lowry et al. (1951). Membrane pellets were stored for up to 1 week at −70°C.

[125I]hα-CGRP Binding Kinetics.

The association and dissociation rates of [125I]hα-CGRP binding to CGRP receptors were determined in membranes prepared from either porcine coronary arteries or from HEK 293 cells expressing the porcine CGRP1 receptor. Frozen membrane pellets were rehomogenized in ice-cold binding buffer (50 mM Tris-HCl, 5 mM MgCl2, 100 mM NaCl, 0.2% bovine serum albumin, and 0.1% bacitracin, pH 7.4) to a concentration of 50 to 100 μg of membrane protein/150 μl. Membrane protein homogenate (150 μl) was added to 13 × 100-mm glass test tubes pretreated with Sigmacote. Fifty microliters of ice-cold binding buffer was added to each test tube, followed by 50 μl of 200 pM [125I]hα-CGRP. To measure the association rate, tubes were quickly vortexed and incubated at 37°C for various times. Bound and free [125I]hα-CGRP were separated by vacuum filtration by pouring the tube contents through Whatman (Maidstone, UK) GF/B glass microfiber filters that were presoaked in 0.2% polyethyleneimine for 30 min. Each tube was rinsed three times with 5 ml of buffer B, and this buffer was also poured through the filter. To measure the dissociation rate, tubes were vortexed and incubated at 37°C for 30 min. Fifty microliters of 5 μM nonradiolabeled hα-CGRP was added to each tube, and the tubes were incubated at 37°C for various times. Bound and free [125I]hα-CGRP were separated by vacuum filtration as described above. The filters were carefully transferred into 12 × 75-mm polyethylene tubes and bound [125I]hα-CGRP was measured in a Wallac Gammamaster 1277 (Gaithersburg, MD) γ-counter.

Competition Binding Assay.

The radioligand binding assay used in this study has been previously described in detail (Abel et al., 1997). Frozen membrane pellets were rehomogenized in ice-cold binding buffer to a concentration of 50 to 100 μg of membrane protein/150 μl. Membrane protein (150 μl) homogenate was added to 13 × 100-mm glass test tubes pretreated with Sigmacote. The tubes were incubated in a 37°C shaking water bath for 50 min in the presence of 40 pM [125I]hα-CGRP and various concentrations of nonlabeled ligands. The total incubation volume was 250 μl. Nonspecific binding was determined using 1 μM hα-CGRP. Whatman GF/B glass microfiber filters were soaked in 0.2% polyethyleneimine for 30 min prior to their use. Bound and free [125I]hα-CGRP were separated by trapping the membranes on the filters and washing with 15 ml of buffer B using a Brandel MB-48R cell harvester (Gaithersburg, MD). Bound [125I]hα-CGRP was measured as described above.

Data Analysis.

Association and dissociation rates of [125I]hα-CGRP binding to CGRP receptors were determined by nonlinear regression analysis using the equations for exponential association and exponential decay. These calculations were performed using GraphPad Prism (San Diego, CA).

Three to five competition binding curves were performed in duplicate for each ligand. Specific binding was determined by subtracting nonspecific binding (defined using 1 μM hα-CGRP) from total binding, and the IC50 for each competition curve was determined by nonlinear regression analysis using GraphPad Prism. The data were fit to both a one-site and a two-site binding model, and the best fit model was determined using an F test. Hill slopes were calculated from nonlinear regression analysis using a sigmoid curve fit model. In kinetic studies, the Kd of [125I]hα-CGRP was 40 pM in membranes prepared from HEK 293 cells expressing the CGRP1 receptor and 14 pM in membranes prepared from porcine coronary arteries. Therefore, these values were used to convert IC50values to KI values by the Cheng-Prusoff equation. Mean pKIvalues for each ligand were compared using a two-tailed Student'st test to determine whether the pKI values in HEK 293 cells were significantly different (p < 0.05) from the pKI values in porcine coronary arteries.

Correlation plots were used to compare antagonist affinities (pKI) determined by competition binding experiments with antagonist affinities determined by their ability to inhibit CGRP-induced relaxation of isolated coronary arteries (pKB). GraphPad Prism was used to perform linear regression analysis, to determine the confidence interval of the correlation, and to calculate the slope and correlation coefficient of these data.

Results

Identification of CRLR and RAMP 1 mRNA in Porcine Coronary Arteries.

Primers that spanned a 223-nucleotide segment of the CRLR mRNA (primers 2 and 4 in Fig. 1) were initially used to search for CRLR mRNA in porcine coronary artery. Porcine lung, the tissue from which this cDNA was originally cloned (Elshourbagy et al., 1998) was used as a positive control. A 223-base pair RT-PCR product was identified in both the porcine coronary artery and lung (Fig.2A), and DNA sequence analysis demonstrated that this product encoded a portion of the porcine CRLR. To confirm that the entire coding region of the CRLR mRNA was present in the coronary artery, we used primers 1 and 3, and primers 2 and 5 (Fig. 1). RT-PCR products from each primer pair were subcloned and sequenced, and the entire nucleotide sequence was submitted to GenBank (GenBank accession number AF419317). The amino acid sequence encoded by this mRNA is identical to that previously reported byElshourbagy et al. (1998). The coding sequence of this mRNA is 1389 nucleotides long and shares 92% and 85% sequence identity with its human and rat orthologs, respectively (accession numbers L76380 andX70658).

Figure 2
Figure 2

Identification of CRLR (A) and RAMP 1 (B) mRNA in porcine coronary artery by RT-PCR. Porcine lung was used as a positive control for the detection of CRLR mRNA. The inclusion/exclusion of reverse transcriptase in the reverse transcription reaction is indicated by ±RT. Marker bands are 200- and 300-base pair size markers, and arrows indicate the 223- and 236-base pair RT-PCR products obtained using primers specific for CRLR (A) and RAMP 1 (B), respectively.

RAMP 1 is an accessory protein that is reportedly required for intracellular trafficking and maturation of the CRLR into the CGRP1 receptor (McLatchie et al., 1998). RAMP 1 mRNA has been previously identified in several human tissues and cell lines, including HEK 293 cells (McLatchie et al., 1998). However, RAMP 1 has not been previously identified in coronary arteries. Since antibodies for this protein were unavailable, we used RT-PCR to identify RAMP 1 mRNA in porcine coronary arteries with the primers described above (Fig. 2B). Porcine RAMP 1 shares 78%, 78%, and 82% nucleotide sequence identity with its rat, mouse, and human orthologs, respectively (GenBank accession numbers AJ001014, AF146522, andAF181550).

Kinetics of [125I]hα-CGRP Binding to CGRP Receptors.

The Kd of [125I]hα-CGRP was determined in membranes from porcine coronary arteries and from porcine CGRP1 receptor-transfected HEK 293 cells by independently measuring the association and dissociation rates of [125I]hα-CGRP binding to CGRP receptors. The procedure for calculating Kd values from kinetic experiments has been described in detail by Limbird (1996). [125I]hα-CGRP dissociated from membranes prepared from porcine CGRP1receptor-transfected HEK 293 cells and from porcine coronary arteries with dissociation rate constants of 0.38/min and 0.46/min, respectively. A representative dissociation curve is shown for CGRP1 receptor-transfected HEK 293 cells in Fig.3A. The association rate constant of [125I]hα-CGRP was also determined, and a representative association curve is shown in Fig. 3B.Kobs values (0.76/min and 1.8/min in HEK 293 cells and coronary arteries, respectively) were converted to association rate constants (9.5 × 109/M/min and 3.4 × 1010/M/min for CGRP1 receptor-transfected HEK 293 cells and coronary arteries, respectively) by the formulaKassoc = (Kobs −Kdissoc)/[radioligand]. The calculated Kd value (Kdissoc/Kassoc) was 40 pM in HEK 293 cells expressing the porcine CGRP1 receptor and 14 pM in coronary arteries. These values are similar to Kd values previously reported for [125I]hα-CGRP in saturation binding experiments using HEK 293 cells stably transfected with the porcine (Kd = 38 pM) or human (Kd = 19 pM) CGRP1 receptor (Aiyar et al., 1996; Elshourbagy et al., 1998).

Figure 3
Figure 3

Representative dissociation (A) and association (B) curves for [125I]hα-CGRP determined in HEK 293 cells expressing the porcine CGRP1 receptor. Similar curves were obtained in membranes prepared from porcine coronary arteries. The association equilibrium constant (Kassoc) was calculated by the equation: Kassoc= (Kobs −Kdissoc)/[radioligand]. The binding equilibrium dissociation constant (Kd) was calculated by the equation: Kd =Kdissoc/Kassoc.

Binding of CGRP Receptor Ligands to CGRP1 Receptors in HEK 293 Cells.

[125I]hα-CGRP was used to label CGRP receptors as previously described (Abel et al., 1997). Specific binding was >90% in membranes from CGRP1 receptor-transfected HEK 293 cells, and maximal inhibition of [125I]hα-CGRP binding for each ligand (except calcitonin) was not different from the maximal inhibition caused by 1 μM hα-CGRP. hα-CGRP and the CGRP1 receptor-selective ligand hα-CGRP8–37 bound with high affinity to membranes from HEK 293 cells stably expressing the CGRP1 receptor (Fig. 4 A; Table 1). The affinity of hα-CGRP8–37 was increased 4-fold by acetylation and 79-fold by benzoylation of the amino terminus. In contrast, the affinity of hα-CGRP8–37 was dramatically decreased by replacing the phenylalanine at position 37 with either alanine ([Ala37]hα-CGRP8–37) or cyclohexylalanine ([Cha37]hα-CGRP8–37). [Pro14]hα-CGRP, a putative CGRP2 receptor-selective ligand, bound with relatively high affinity. The prototypical CGRP2-selective agonist [Cys(ACM)2,7]hα-CGRP bound to the CGRP1 receptor with 400-fold lower affinity than hα-CGRP. Adrenomedullin, another member of the CGRP peptide family, competed for [125I]hα-CGRP binding sites with 248-fold lower affinity than hα-CGRP, and calcitonin (1 nM–1 μM) did not compete at all. Competition binding curves for several of these ligands are shown in Fig. 4A, and meanKI values for all ligands are listed in Table 1. Competition curves for each ligand (except calcitonin) fit best to a single-site binding model.

Figure 4
Figure 4

Mean competition binding curves for selected ligands at porcine CGRP receptors. A, ligand inhibition of [125I]hα-CGRP binding in membranes from HEK 293 cells expressing the cloned porcine CGRP1 receptor. B, ligand inhibition of [125I]hα-CGRP binding in membranes from porcine coronary arteries. Curves represent the mean of three to five individual experiments each using cells grown in different cell culture flasks or using porcine coronary arteries from different animals.

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Table 1

KI values of CGRP analogs in pig coronary arteries and in pig CGRP1 receptor-transfected HEK 293 cells

Binding of CGRP Receptor Ligands to CGRP Receptors in Porcine Coronary Arteries.

Specific binding was >70% in all experiments using membranes from porcine coronary arteries, and maximal inhibition of [125I]hα-CGRP binding for each ligand (except calcitonin) was not different from the maximal inhibition caused by 1 μM hα-CGRP. KI values were determined for the prototypical CGRP1 and CGRP2 receptor-selective ligands {hα-CGRP8–37 and [Cys(ACM)2,7]hα-CGRP, respectively} as well as several other peptides. Previous studies have demonstrated that the hα-CGRP8–37 derivatives used in this investigation inhibit CGRP-induced relaxation of porcine coronary arteries with KB values ranging from 29 nM to >200 μM (Saha et al., 1998; Smith et al., 2001; D. J. J. Waugh, personal communication). Therefore, we characterized the CGRP receptors in coronary arteries with ligands that were predicted to bind with a broad range of affinities. hα-CGRP and hα-CGRP8–37 bound to membranes from porcine coronary arteries with high affinity (0.11 and 6.6 nM, respectively). Consistent with previous studies (Smith et al., 2001), the affinity of hα-CGRP8–37 was increased 5-fold by acetylation and 24-fold by benzoylation of the amino terminus. In contrast, the affinity of hα-CGRP8–37 was reduced by replacing the phenylalanine at position 37 with alanine or cyclohexylalanine (Table 1). The CGRP2receptor-selective peptide [Cys(ACM)2,7]hα-CGRP bound with a 349-fold lower affinity than hα-CGRP and a 6-fold lower affinity compared with hα-CGRP8–37. The high-affinity binding of hα-CGRP8–37 and lower-affinity binding of [Cys(ACM)2,7]hα-CGRP is consistent with the presence of the CGRP1 receptor in this tissue. Adrenomedullin competed for [125I]hα-CGRP binding sites with 180-fold lower affinity than hα-CGRP, and calcitonin (1 nM–1 μM) did not compete for [125I]hα-CGRP binding sites at all. Competition binding curves for several of these ligands are shown in Fig. 4B, and mean KI values for all ligands are listed in Table 1. Competition curves for all ligands (except calcitonin) fit best to a single binding site model.

Comparison of Ligand Affinities for CGRP Receptors in Porcine Coronary Arteries and HEK 293 Cells.

A comparison of ligand affinities in membranes from porcine coronary arteries and HEK 293 cells expressing the porcine CGRP1 receptor is shown in Table 1. There were no significant differences (p < 0.05) for any of these peptides when comparing their affinities for the CGRP receptors in porcine coronary arteries with their affinities for porcine CGRP1 receptors transfected into HEK 293 cells. In addition, pKI values in porcine coronary arteries and porcine CGRP1 receptor-transfected HEK 293 cells demonstrated a strong correlation (r2 = 0.99) (Fig.5A).

Figure 5
Figure 5

Correlation plots of affinity values determined by radioligand binding (pKI) or isolated tissue experiments (pKB). A, the correlation between antagonist affinities determined by radioligand binding in coronary arteries and HEK 293 cells expressing the porcine CGRP1 receptor. B, the correlation between antagonist affinities determined by radioligand binding or isolated tissue experiments with porcine coronary arteries. C, the correlation between antagonist affinities determined by radioligand binding in HEK 293 cells expressing the porcine CGRP1 receptor and antagonist affinities determined by isolated tissue experiments in porcine coronary arteries. The solid line is the linear regression line calculated from the data points; the dotted lines represent the 95% confidence interval of the linear regression; and the dashed line represents the line of identity. The slope and the correlation coefficient (r2) of the linear regression of the data points is also indicated.

Antagonist affinities (KI) determined by competition binding were 22- to 707-fold higher than the affinities that we have previously observed for these ligands in functional assays that measure their inhibition of CGRP-induced relaxation of isolated porcine coronary arteries (Table 2). Therefore, we examined the correlation between the affinity values determined by functional assays with coronary arteries (pKB) and the affinities determined by competition binding using membranes (pKI) from the same tissue. The affinity of each antagonist was higher when measured by radioligand binding than when measured in functional assays. Thus the linear regression lines correlating these data are not superimposed with the line of identity (Fig. 5 B). However, there was a high correlation (r2 = 0.88) between affinity values determined by the two methods. There was also a high correlation (r2 = 0.86) between radioligand binding affinities in CGRP1 receptor-transfected HEK 293 cells and affinities determined by functional experiments with isolated coronary arteries (Fig. 5C). For both correlations (Fig. 5, B and C), the 95% confidence interval of the slope of the regression line included the value of 1.0.

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Table 2

Comparison of ligand affinities determined by competition binding (KI) or isolated tissue experiments (KB) in pig coronary arteries

Discussion

CGRP receptors have been classified into CGRP1 and CGRP2 receptor subtypes based upon their affinity for hα-CGRP8–37 in isolated tissue experiments. hα-CGRP8–37 is an antagonist that inhibits CGRP1 receptor-mediated responses with high affinity (KB < 100 nM) and inhibits putative CGRP2 receptor-mediated responses with low affinity (KB > 1 μM) (Dennis et al., 1990). In addition, [Cys(ACM)2,7]hα-CGRP has been proposed to be an agonist at CGRP2receptors and inactive at CGRP1 receptors (Dennis et al., 1989). In isolated tissue studies, Foulkes et al. (1991) have previously reported that CGRP receptors in large porcine coronary arteries (outside diameter >1 mm) have low affinity for hα-CGRP8–37(KB > 1 μM). Our laboratory has also found that hα-CGRP8–37 has low affinity (KB > 1 μM) in large porcine coronary arteries and that [Cys(ACM)2,7]hα-CGRP causes relaxation of this tissue (Waugh et al., 1999). These functional studies have established large coronary arteries as a model for studying the CGRP2 receptor.

In contrast to previous studies, we report two independent lines of evidence to show that porcine coronary arteries express the CGRP1 receptor. First, we have identified mRNA encoding CRLR and RAMP 1 in porcine coronary arteries. These proteins have been previously shown to form the CGRP1receptor (McLatchie et al., 1998). Second, we have found that CGRP1 and CGRP2receptor-selective ligands do not discriminate between CGRP receptors in porcine coronary arteries and porcine CGRP1receptors that have been transfected into HEK 293 cells. Furthermore, correlations between affinity values determined by radioligand binding and by isolated tissue experiments suggest that the CGRP receptor that has low affinity for hα-CGRP8–37 in functional studies with coronary arteries is the same CGRP receptor that has been cloned and expressed in HEK 293 cells. These data do not support the current view that the CGRP1 and CGRP2 receptor subtypes are different proteins that represent independent receptors.

We used radioligand binding to compare the affinity of several ligands for CGRP receptors expressed in coronary arteries with their affinities for CGRP1 receptors expressed in HEK 293 cells. Stable expression of the porcine CGRP1 receptor in HEK 293 cells provides a system that is free of other receptors that might bind CGRP and its analogs. This also allowed the comparison of CGRP receptor subtypes from the same species. This is important because many other comparisons of CGRP1 and CGRP2 receptors have been complicated by the use of tissues from different species to represent the putative CGRP receptor subtypes. We found that the affinity of hα-CGRP8–37, the prototypical CGRP1 receptor-selective antagonist, and of [Cys(ACM)2,7]hα-CGRP, the putative CGRP2 receptor-selective agonist, were nearly identical in membranes prepared from porcine coronary arteries and from porcine CGRP1 receptor-transfected HEK 293 cells. In fact, each of the 12 ligands used in the radioligand binding assay bound to a single binding site in the porcine coronary artery, and none of the ligands were capable of discriminating the cloned CGRP1 receptor from the CGRP receptor that is found in porcine coronary arteries. These data provide evidence that porcine coronary arteries express only the CGRP1receptor.

In contrast to previous functional studies with isolated coronary arteries, the radioligand binding data in the present investigation demonstrate that hα-CGRP8–37 has a CGRP1 receptor-like affinity for CGRP receptors in porcine coronary arteries. This raised the possibility that the CGRP receptor identified by radioligand binding is not the same CGRP receptor that has been characterized by isolated tissue experiments. Therefore, we used correlation plots to compare the antagonist affinities determined by isolated tissue experiments (KB) with their affinities determined by radioligand binding (KI). Each antagonist demonstrated a lower affinity in functional experiments with isolated coronary arteries than in radioligand binding experiments with membranes prepared from the same tissue. However, correlation plots demonstrated that the difference betweenKB andKI values was consistent for each ligand, and that there was a high correlation between affinity values determined by the two different methods. Regardless of the method used to measure ligand affinities in porcine coronary arteries, there was also a high correlation between affinity values in this tissue and in membranes from CGRP1 receptor-transfected HEK 293 cells. These data support the conclusion that isolated tissue experiments and competition binding experiments with porcine coronary arteries both identify the same CGRP receptor.

Our radioligand binding data raise an important question: Why does hα-CGRP8–37 appear to identify a low-affinity CGRP2 receptor in functional assays with isolated tissue but not in competition binding experiments? One explanation for the low affinity of hα-CGRP8–37 in isolated porcine coronary arteries, rat vas deferens, and other tissues is that this ligand may be degraded by proteases, causing the peptide to appear to have a lower affinity in these tissues than in tissues that lack these enzymes. Fernandez-Patron et al. (2000) reported that matrix metalloprotease-2, a protease present in vascular smooth muscle and endothelium, specifically cleaves hβ-CGRP into hβ-CGRP1–14 and hβ-CGRP15–37. In addition, we found that acetylation of the amino terminus of hα-CGRP8–37, a modification that has been demonstrated to protect other peptides from degradation (Drapeau et al., 1993), caused a 160-fold increase in the affinity (KB) of this ligand in functional relaxation assays with porcine coronary arteries (Smith et al., 2001). In contrast, acetylation of hα-CGRP8–37 caused only a 5-fold increase in its binding affinity (KI) for membranes from the same tissue. These data suggest that hα-CGRP8–37may be more susceptible to proteolytic degradation in whole coronary arteries than in membranes. Two nonpeptide CGRP receptor ligands (SB-273779 and BIBN4096BS) have recently been developed (Doods et al., 2000; Aiyar et al., 2001) and may be useful for avoiding ligand degradation while studying CGRP receptors.

A disadvantage of radioligand binding experiments using membranes is that the receptor is removed from its native environment and placed under conditions that may cause receptor accessory proteins to be lost. We found that hα-CGRP8–37 has a low, CGRP2 receptor-like affinity in functional studies of isolated porcine coronary arteries and a high, CGRP1 receptor-like affinity in membrane binding assays. One explanation for this difference is that the binding characteristics of the CGRP1 receptor are modified during the membrane preparation procedure. McLatchie et al. (1998) and Evans et al. (2000) have reported that RAMP 1 and receptor component protein (RCP) are required to form a functional CGRP1 receptor. RAMP 1 is an integral membrane protein with a membrane-spanning domain that presumably protects it from being lost during the membrane preparation procedure. However, RCP is a peripheral membrane protein that can be dissociated from the membrane (Evans et al., 2000). Although the affinity of hα-CGRP is unaffected by the presence or absence of RCP (Evans et al., 2000), the effect of this protein on hα-CGRP8–37 has not been examined. It is possible that low-affinity hα-CGRP8–37 binding is conferred by RCP in intact tissues and that hα-CGRP8–37 does not have low affinity in competition binding experiments because RCP is lost during the membrane preparation procedure.

The binding affinities of CGRP receptor agonists were also determined in our study. Previous investigators have reported that [Cys(ACM)2,7]hα-CGRP and [Pro14]hα-CGRP are selective for the putative CGRP2 receptor (Dennis et al., 1989; Li et al., 1997). In contrast, [Cys(ACM)2,7]hα-CGRP and [Pro14]hα-CGRP demonstrated no selectivity for CGRP receptors in coronary arteries over CGRP1 receptors in HEK 293 cells in our competition binding experiments. Furthermore, we have found that the putative CGRP2 receptor agonist, [Pro14]hα-CGRP, stimulates 3′,5′ cyclic adenosine monophosphate production (EC50 = 158.7 ± 113.2 nM) in porcine CGRP1receptor-transfected HEK 293 cells (B. R. Rorabaugh, P. W. Abel, D. D. Smith, and M. S. Scofield, unpublished data). The ability of these ligands to demonstrate agonist activity in some CGRP1 receptor systems and not in others suggests that the selectivity of these ligands is caused by something other than the presence of a second CGRP receptor subtype. One possibility is that the tissue to tissue variation in potency of these agonists is caused by different amounts of receptor reserve. We have previously shown that [Cys(ACM)2,7]hα-CGRP is a partial agonist in porcine coronary arteries (Waugh et al., 1999). This is consistent with the presence of a CGRP1 receptor reserve in tissues in which [Cys(ACM)2,7]hα-CGRP is an agonist and an absence of a CGRP1 receptor reserve in tissues in which this ligand shows no agonist activity (Kenakin, 1993). Therefore, the existence of a second CGRP receptor subtype is not necessary to explain the ability of [Cys(ACM)2,7]hα-CGRP and [Pro14]hα-CGRP to be agonists in some isolated tissues and inactive in others.

In summary, CGRP receptors in porcine coronary arteries have been previously classified as the CGRP2 receptor subtype because hα-CGRP8–37 antagonizes CGRP-induced relaxation of these arteries with low affinity. However, we have demonstrated that porcine coronary arteries have CRLR and RAMP 1 mRNA and that the ligand binding characteristics of CGRP receptors in porcine coronary arteries are identical to those of the cloned porcine CGRP1 receptor. Furthermore, the correlation between KB andKI values are consistent with the conclusion that the low affinity (KB> 1 μM) of hα-CGRP8–37 in functional studies using isolated porcine coronary arteries occurs at the CGRP1 receptor. Our data do not support the idea that CGRP1 and CGRP2receptors represent two independent proteins with different affinities for the antagonist, hα-CGRP8–37. Rather, our results suggest that there is only one CGRP receptor that can have different affinities for hα-CGRP8–37 in functional studies, based upon various tissue-dependent factors.

Acknowledgments

We thank Joe Haun of J & J Quality Meats (Elkhorn, NE) and Al Lieberum of Hormel Foods Corporation (Fremont, NE) for providing porcine tissues. HEK 293 cells stably expressing the porcine CGRP1 receptor were a gift from Dr. Allan R. Shatzman at SmithKline Beecham.

Footnotes

  • Supported by Grant HL51131 from the National Institutes of Health, the State of Nebraska Cancer and Smoking Related Diseases Research Program, and the Health Future Foundation.

  • Portions of this work have been previously published in abstract form: Rorabaugh B, Abel P, Smith D, and Scofield M (2000) Evidence for the CGRP1 receptor in porcine coronary artery. FASEB J14:A1404.

  • Abbreviations:
    CGRP
    calcitonin gene-related peptide
    Cha
    cyclohexylalanine
    CRLR
    calcitonin receptor-like receptor
    CGRP1
    calcitonin gene-related peptide subtype 1
    CGRP2
    calcitonin gene-related peptide subtype 2
    hα-CGRP
    human α-calcitonin gene-related peptide
    HEK
    human embryonic kidney
    [125I]hα-CGRP
    2-[125I]iodohistidyl10hα-CGRP
    IC50
    ligand concentration that inhibits 50% of the radioligand bound at equilibrium
    KB
    functional equilibrium dissociation constant
    Kd
    kinetic equilibrium dissociation constant
    KI
    binding equilibrium dissociation constant
    Kassoc
    association rate constant
    Kobs
    observed association rate constant
    Kdissoc
    dissociation rate constant
    PCR
    polymerase chain reaction
    RAMP 1
    receptor activity modifying protein 1
    RCP
    receptor component protein
    RT-PCR
    reverse transcription-polymerase chain reaction
    • Received June 29, 2001.
    • Accepted August 28, 2001.

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Research ArticleCARDIOVASCULAR

Functional Calcitonin Gene-Related Peptide Subtype 2 Receptors in Porcine Coronary Arteries Are Identified as Calcitonin Gene-Related Peptide Subtype 1 Receptors by Radioligand Binding and Reverse Transcription-Polymerase Chain Reaction

Boyd R. Rorabaugh, Margaret A. Scofield, D. David Smith, William B. Jeffries and Peter W. Abel
Journal of Pharmacology and Experimental Therapeutics December 1, 2001, 299 (3) 1086-1094;
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