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Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) receptors and their respective ligands play important roles in cardiovascular (patho-)physiology. Functional expression of ADM and CGRP receptors requires the presence of the calcitonin receptor-like receptor (CRLR) together with receptor-activity-modifying proteins (RAMPs). We have characterized the expression patterns of CRLR and RAMP1 to RAMP3 in human cardiovascular-related tissues by quantitative polymerase chain reaction. We could identify high expression levels of CRLR, RAMP1, and RAMP2 in human heart and various blood vessels. RAMP3 expression in these tissues, however, was detectable at significantly lower levels. In addition, we describe here a novel, aequorin luminescence-based G protein-coupled receptor reporter assay that enables the real-time detection of receptor activation in living cells. In the assay system, intracellular cAMP levels are monitored with high sensitivity by using a modified, heteromultimeric cyclic nucleotide-gated channel mediating calcium influx. G(q)-coupled receptor activation is detected via aequorin luminescence stimulated by calcium release from intracellular stores. Using this novel reporter assay, we established and characterized stable ADM1 and CGRP1 receptor cell lines. The peptide ligands ADM, CGRP1, and CGRP2 were characterized as potent agonists at their respective receptors. In contrast, intermedin acted as a weak agonist on both receptors and showed only partial agonism on the ADM1 receptor. Agonist activities were effectively antagonized by the receptor antagonists ADM(22-52) and CGRP(8-37). Various vasoactive ADM fragments were also characterized but showed no activity on the ADM1 receptor cell line. In addition, luminescence signal kinetics after activation of G(s)- and G(q)-coupled receptors were found to be markedly different.

Calcitonin receptors (CTRs) dimerize with receptor activity-modifying proteins (RAMPs) to generate high-affinity amylin (AMY) receptors; however, the relative contribution of individual RAMP domains to the formation of AMY receptors is poorly understood. We have used chimeras between RAMP1 and RAMP2 that specifically exchanged the N-terminal, transmembrane, or C-terminal domain and examined these in assays of [(125)I]amylin binding or peptide-induced cAMP signaling in COS-7 cells transiently transfected with wild-type or chimeric RAMPs and human CTRa. The specificity of peptides in competition for [(125)I]amylin binding was principally dictated by the N-terminal domain present in the chimeras; however, the maximal level of binding induced was dictated by the transmembrane domain present. This extended previous data (Zumpe et al., 2000) to provide a distinction between the transmembrane domain and the C terminus in this function. In contrast to the effects on binding, each of the RAMP domains played a role in the signaling phenotype of the receptors. In particular, the potency of calcitonin gene-related peptide (CGRP) was most influenced by the C-terminal domain present, in which the presence of the RAMP1 C-terminal domain led to increased potency over CTRa alone, whereas chimeras with the RAMP2 C-terminal domain did not induce increased CGRP potency. The data provide additional support for the importance of the N terminus in determining binding affinity but reveal a prominent role of the transmembrane domain in the strength of amylin binding and a unique role for the C terminus in signaling by peptides to stimulate cAMP production.

Calcitonin (CT) receptors dimerize with receptor activity-modifying proteins (RAMPs) to create high-affinity amylin (AMY) receptors, but there is no reliable means of pharmacologically distinguishing these receptors. We used agonists and antagonists to define their pharmacology, expressing the CT(a) receptor alone or with RAMPs in COS-7 cells and measuring cAMP accumulation. Intermedin short, otherwise known as adrenomedullin 2, mirrored the action of alpha CGRP, being a weak agonist at CT(a), AMY(2a), and AMY(3a) receptors but considerably more potent at AMY(1a) receptors. Likewise, the linear calcitonin gene-related peptide (CGRP) analogs (Cys(ACM)(2,7))h alpha CGRP and (Cys(Et)(2,7))h alpha CGRP were only effective at AMY(1a) receptors, but they were partial agonists. As previously observed in COS-7 cells, there was little induction of the AMY(2a) receptor phenotype; thus, AMY(2a) was not examined further in this study. The antagonist peptide salmon calcitonin(8-32) (sCT(8-32)) did not discriminate strongly between CT and AMY receptors; however, AC187 was a more effective antagonist of AMY responses at AMY receptors, and AC413 additionally showed modest selectivity for AMY(1a) over AMY(3a) receptors. CGRP(8-37) also demonstrated receptor-dependent effects. CGRP(8-37) more effectively antagonized AMY at AMY(1a) than AMY(3a) receptors, although it was only a weak antagonist of both, but it did not inhibit responses at the CT(a) receptor. Low CGRP(8-37) affinity and agonism by linear CGRP analogs at AMY(1a) are the classic signature of a CGRP2 receptor. Our data indicate that careful use of combinations of agonists and antagonists may allow pharmacological discrimination of CT(a), AMY(1a), and AMY(3a) receptors, providing a means to delineate the physiological significance of these receptors.

Coexpression of receptor activity-modifying proteins (RAMPs) with calcitonin receptor 2 (CTR2) or calcitonin receptor-like receptor (CRLR) leads to the formation of four functional heterodimeric receptors for human calcitonin gene-related peptide (hCGRP). In this study, we transfected hCGRP receptors into human embryonic kidney 293 cells and examined their pharmacological profiles using three dominant-negative (DN) RAMP mutants and various hCGRPalpha analogs. Fluorescence-activated cell-sorting analysis revealed that their cotransfection with CTR2 induced cell surface expression of all three RAMPs, and the three CTR2/RAMP heterodimers mediated equivalent levels of cAMP production in response to hCGRPalpha that were approximately 50-fold greater than were seen with CTR2 alone. By contrast, [Tyr0]hCGRPalpha binding and signaling were markedly weaker with CTR2/RAMP2 or -3 than with CTR2/RAMP1 or CRLR/RAMP1; likewise, 125I-[His10]hCGRPalpha bound most potently to CTR2/RAMP1. When CTR2 was coexpressed with DN RAMP1 or -2, hCGRPalpha-evoked responses were similar to those seen with CTR2 alone, despite the expression of both CTR2 and DN RAMP at the cell surface. But coexpression of DN RAMP3 with CTR2 significantly diminished hCGRPalpha signaling compared with that seen with CTR2 alone, indicating that DN RAMP3 is able to function as a negative regulator of CTR2 function. Competition experiments showed the relative agonist sensitivity of the four receptors to be hCGRPalpha > [Tyr0]hCGRPalpha > [Cys(Et)2,7]hCGRPalpha > [Cys(ACM)2,7]hCGRPalpha. Of the linear analogs, [Cys(ACM)2,7]hCGRPalpha (ACM, acetylmethoxy) enhanced cAMP formation only via CTR2/RAMP1, whereas [Cys(Et2,7)]hCGRPalpha acted via CRLR/RAMP1 and somewhat less potently via CTR2/RAMP1. Thus, among the three CGRP8-37-insensitive receptors, CTR2/RAMP1 is most sensitive to the two linear analogs, suggesting that it could be classified as a CGRP2 receptor. Moreover, the combined use of iodinated CGRPalpha analogs may be useful for defining the CGRP1 receptor.

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and amylin are structurally related peptides mediating vasorelaxation in the coronary circulation possibly via CGRP receptors (subtypes 1 or 2). Functional CGRP1 receptors appear to consist of at least three different kinds of proteins: the calcitonin receptor-like receptor (CRLR), receptor-activity-modifying proteins (RAMPs) and the receptor component protein (RCP). No CGRP2 receptor has yet been cloned. Using reverse transcriptase - polymerase chain reaction, the presence of mRNA sequences encoding CRLR, RCP and RAMPs was demonstrated in human coronary arteries. Relaxant responses were studied on isolated segments of coronary arteries after precontraction with U46619 (9,11-dideoxy-11alpha,9alpha-epoxymethano-prostaglandin F(2alpha)). The human peptides alphaCGRP, AM, and amylin induced relaxation with mean pEC50 values of 8.6, 6.8, and 6.3 M, respectively. Preincubation with alphaCGRP(8-37) (10(-7) -10(-5) M) and a novel nonpeptide CGRP antagonist "Compound 1" (WO98/11128) (10(-7)-10(-5) M) caused a dose-dependent rightward shift of the concentration-response curves for alphaCGRP with pA(2) values of 7.0 and 7.1, respectively. Preincubation with alphaCGRP(8-37) (10(-6) M) and Compound 1 (10(-6) M) caused significant rightward shift of the concentration-response curves for AM and amylin as well with pK B values between 6.6 and 7.5. Preincubation with AM(22-52) had no antagonistic effect on the AM and amylin response, neither did diacetoamidomethyl cysteine CGRP cause any concentration dependent (10(-11)-10(-6) M) dilatation. In conclusion, mRNA for the components forming CGRP1 and AM receptors was detected in the human left anterior descending coronary arteries. alphaCGRP, AM, and amylin mediated vasorelaxation via the CGRP1 receptor. Compound 1 acted as a nonpeptide antagonist at the CGRP1 receptor and could thus become a tool for the study of CGRP-mediated functional responses in human tissue.

Calcitonin gene-related peptide (CGRP) receptors are classified into CGRP subtype 1 (CGRP(1)) and CGRP subtype 2 (CGRP(2)) based on the affinity of the antagonist, human alpha (halpha)-CGRP(8-37). halpha-CGRP(8-37) antagonizes CGRP(1) receptor-mediated responses with high affinity (K(B) < 100 nM) and antagonizes CGRP(2) receptor-mediated responses with low affinity (K(B) > 1 microM). CGRP(2) receptors have been previously reported to mediate relaxation of large porcine coronary arteries because this action is antagonized with low affinity by halpha-CGRP(8-37). 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 CGRP(1) 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 CGRP(1) receptor were highly similar. K(I) values for halpha-CGRP(8-37) were 6.6 and 5.7 nM in porcine coronary arteries and the cloned CGRP(1) receptor, respectively. The affinities (K(B)) of halpha-CGRP(8-37) 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 halpha-CGRP(8-37) antagonizes CGRP-induced relaxation of porcine coronary arteries with low affinity at the CGRP(1) receptor. Taken together, these data do not support the existence of the CGRP(2) receptor.

The quantitative comparison of the relative potency of agonists is a standard method of receptor and agonist classification. If agonist potency ratios do not correspond in two given tissues, this is used as presumptive data to conclude that the receptors in those two tissues are different. This article presents data to show that a single receptor can demonstrate varying agonist potency ratios in different host cells. These data are described in terms of the production of more than one agonist-selective receptor active state and the interaction of these different active states with multiple G proteins in the membrane to produce cellular response. Stable host human embryonic kidney 293 cells with enhanced quantities of the respective Galpha-protein were created. Wild-type and Galpha-subunit enriched cells were then transiently transfected with human calcitonin receptor type 2 (hCTR2). Binding did not detect differences in the G protein-enriched cells versus wild-type cells. In contrast, functional studies did show differences between the host cell lines and Galpha-subunit enriched cell lines. The relative potency of eight calcitonin agonists was measured in studies of calcium fluorescence in transfected cells containing human calcitonin receptor type 2 by comparing pEC(50) (-log molar concentration producing half-maximal response) values. In Galphas-enriched cells, the relative order of potency of the agonists changed. The host-cell dependent differences in potency ratios ranged from 2-fold to more than 46-fold. This finding is not consistent with the idea that all of the agonists produce response in the same manner (i.e., through a common active state of the receptor). These data are consistent with the idea that these different agonists produce arrays of active states that differentially use G proteins. This idea is discussed in terms of the design of stimulus-bias assay systems to detect agonist-selective receptor active states with resulting potential for increased selectivity of agonists.

This article describes the behavior of transiently transfected human receptors into melanophores and the potential use of constitutive receptor activity to screen for new drug entities. Specifically, transient transfection of melanophores with different concentrations of receptor cDNA presumably leads to increased levels of receptor expression. This leads to an increased response to agonists (both maxima and potency) and, in some cases, an agonist-independent constitutive receptor activity. Transfections with increasing concentrations of the G(s) protein-coupled human calcitonin receptor type 2 (hCTR2) cDNA produced sufficient levels of constitutively activated receptor to cause elevated basal cellular responses. This was observed as a decrease in the transmittance of light through melanophores (consistent with G(s) protein activation) and increased response to human calcitonin. The receptor-mediated nature of this response was confirmed by its reversal with the hCTR2 peptide inverse agonist AC512. A collection of ligands for hCTR2 either increased or decreased constitutive hCTR2 activity, suggesting that the constitutive system was a sensitive discriminator of positive and negative ligand efficacy. Similar results were obtained with G(i)-protein-coupled receptors. Transient transfection of NPY1, NPY2, NPY4, CXCR4, and CCR5 cDNA produced increased light transmittance through melanophores (consistent with G(i)-protein activation). NPY1 cDNA produced little constitutive response on transfection, whereas maximal levels of constitutive activity ranging from 30 to 45% were observed for the other G(i)-protein-coupled receptors. Responses to agonists for these receptors increased (both maxima and potency) with increasing cDNA transfection. The receptor/G(i)-protein nature of both the constitutive and agonist-mediated responses was confirmed by elimination with pertussis toxin pretreatment. These data are discussed in terms of the theoretical aspects of constitutive receptor activity and the applicability of this approach for the general screening of G protein-coupled orphan receptors.

Human breast cell carcinoma MCF-7 cells were found to bind 125I-labeled rat amylin (rAmylin) and the peptide amylin antagonist radioligand 125I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already defined high affinity binding site for amylin in the rat nucleus accumbens [Mol. Pharmacol. 44:493-497 (1993); J. Pharmacol. Exp. Ther. 270:779-787 (1994); Eur. J. Pharmacol. 262:133-141 (1994)]. To further define this receptor, we report results of expression cloning studies from an MCF-7 cell library. We isolated two variants of a seven-transmembrane receptor that were identical to two previously described human calcitonin receptors (hCTR1 and hCTR2). These receptors were characterized by expression in different surrogate host cell systems. Transient expression of hCTR1 in COS cells yielded membranes that bound 125I-AC512 and 125I-salmon calcitonin with high affinity, but no high affinity binding was observed with 125I-human calcitonin (hCAL) or 125I-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCTR2 in COS cells yielded membranes that bound 125I-AC512, 125I-hCAL, and 125I-rAmylin with high affinity. The agonists 125I-hCAL and 125I-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radioligand 125I-AC512 in this expression system. This pattern of binding was repeated in HEK 293 cells stably transfected with hCTR2 (125I-hCAL = 24.8% Bmax, 125I-rAmylin = 8% Bmax). In both expression systems, the agonists hCAL and rAmylin were much more potent in displacing their radioligand counterparts than was the antagonist radioligand 125I-AC512. For example, the pKi value for displacement of 125I-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 when displacing 125I-rAmylin. Finally, hCTR2 was expressed in baculovirus-infected Ti ni cells. In this system, only specific binding to the antagonist 125I-AC512 and agonist 125I-hCAL was observed; no binding to 125I-rAmylin could be detected. These data are discussed in terms of two working hypotheses. The first is that amylin is a weak agonist for hCTR2 and that this receptor is unrelated to the amylin receptor found in this cell line. The second is that hCTR2 couples to different G proteins for calcitonin and amylin function in different cells. At present, these data cannot be used to disprove conclusively either hypothesis.

We have cloned and characterized a second form of the human calcitonin receptor from T47D cells. It resembles the clone described by Gorn et al. [J. Clin. Invest. 90:1726-1735 (1992)] except that it lacks a 16-amino acid insert in the putative first intracellular loop. The insert-negative receptor appears to be the most abundant form, and it occurs at a relatively constant level in all expressing tissues. In contrast, the insert-positive receptor is found at low levels in most tissues but its expression levels appear to be much more variable. The insert-negative cDNA was stably expressed in baby hamster kidney cells. Like the endogenous T47D receptor, the recombinant receptor has an equally high affinity for salmon and porcine calcitonin but a 3-4-fold lower affinity for human calcitonin. High concentrations of calcitonin gene-related peptide, rat amylin, secretin, or vasoactive intestinal peptide do not significantly compete with calcitonin for binding to the recombinant receptor. Calcitonin stimulates a cAMP response in both T47D and transfected baby hamster kidney cells. Salmon calcitonin is more potent than human calcitonin for T47D cells, but the two are nearly equipotent for the transfectants. Furthermore, the ED50 for the cAMP response in the transfectants is 10-100-fold lower than in T47D cells. Calcitonin stimulates inositol phosphate turnover and elevates internal calcium levels in the transfectants. This response requires non-physiological levels of calcitonin and is directly correlated with the number of receptors. Lastly, by using a human/rodent somatic cell hybrid panel and in situ hybridization, we localized the human calcitonin receptor gene to chromosome 7.