David R. Poyner

International Union of Pharmacology. XXXII. The Mammalian Calcitonin Gene-Related Peptides, Adrenomedullin, Amylin, and Calcitonin Receptors

The calcitonin family of peptides comprises calcitonin, amylin, two calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first calcitonin receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The receptors for the other members the family are made up of subunits. The calcitonin-like receptor (CL receptor) requires a single transmembrane domain protein, termed receptor activity modifying protein, RAMP1, to function as a CGRP receptor. RAMP2 and -3 enable the same CL receptor to behave as an adrenomedullin receptor. Although the calcitonin receptor does not require RAMP to bind and respond to calcitonin, it can associate with the RAMPs, resulting in a series of receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric receptors.

Calcitonin gene-related peptide: Multiple actions, multiple receptors

Calcitonin gene-related peptide (CGRP) shows diversity both in its effects and its receptors. It is likely to have roles as a neurotransmitter, neuromodulator, local hormone and trophic factor. Its effects include rapid changes in neuronal activity, relaxation of many types of smooth muscle, actions on metabolism and changes in gene expression. Receptor heterogeneity has been revealed from experiments comparing agonist potency ratios and antagonist affinities. The evidence from these approaches is reviewed in this article and a speculative receptor classification scheme is proposed. Some of the likely future directions for CGRP research are discussed.

Pharmacology of receptors for calcitonin gene-related peptide and amylin

Calcitonin gene-related peptide (CGRP), a widespread neuropeptide with multiple actions, has substantial homology with amylin, a peptide implicated in insulin-resistant diabetes, and adrenomedullin, a recently discovered potent vasodilator. There is controversy over the existence of CGRP receptor subtypes, and whether independent receptors exist for amylin and adrenomedullin. In this article, the current status of CGRP receptor classification is reviewed by David Poyner, taking particular account of species differences, and evidence is presented supporting the existence of multiple receptors for CGRP, as well as independent binding sites for amylin.

CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22–52, CGRP8–37 and BIBN4096BS

Adrenomedullin (AM) has two known receptors formed by the calcitonin receptor‐like receptor (CL) and receptor activity‐modifying protein (RAMP) 2 or 3: We report the effects of the antagonist fragments of human AM and CGRP (AM22–52 and CGRP8–37) in inhibiting AM at human (h), rat (r) and mixed species CL/RAMP2 and CL/RAMP3 receptors transiently expressed in Cos 7 cells or endogenously expressed as rCL/rRAMP2 complexes by Rat 2 and L6 cells. AM22–52 (10 μM) antagonised AM at all CL/RAMP2 complexes (apparent pA2 values: 7.34±0.14 (hCL/hRAMP2), 7.28±0.06 (Rat 2), 7.00±0.05 (L6), 6.25±0.17 (rCL/hRAMP2)). CGRP8–37 (10 μM) resembled AM22–52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent pA2 values: 7.04±0.13 (hCL/hRAMP2), 6.72±0.06 (Rat2), 7.03±0.12 (L6)). On CL/RAMP3 receptors, 10 μM CGRP8–37 was an effective antagonist at all combinations (apparent pA2 values: 6.96±0.08 (hCL/hRAMP3), 6.18±0.18 (rCL/rRAMP3), 6.48±0.20 (rCL/hRAMP3)). However, 10 μM AM22–52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA2 6.73±0.14). BIBN4096BS (10 μM) did not antagonise AM at any of the receptors. Where investigated (all‐rat and rat/human combinations), the agonist potency order on the CL/RAMP3 receptor was AM∼βCGRP>αCGRP. rRAMP3 showed three apparent polymorphisms, none of which altered its coding sequence. This study shows that on CL/RAMP complexes, AM22–52 has significant selectivity for the CL/RAMP2 combination over the CL/RAMP3 combination. On the mixed species receptor, CGRP8–37 showed the opposite selectivity. Thus, depending on the species, it is possible to discriminate pharmacologically between CL/RAMP2 and CL/RAMP3 AM receptors.

Lifting The Lid On G-Protein-Coupled Receptors: The Role Of Extracellular Loops.

GPCRs exhibit a common architecture of seven transmembrane helices (TMs) linked by intracellular loops and extracellular loops (ECLs). Given their peripheral location to the site of G‐protein interaction, it might be assumed that ECL segments merely link the important TMs within the helical bundle of the receptor. However, compelling evidence has emerged in recent years revealing a critical role for ECLs in many fundamental aspects of GPCR function, which supported by recent GPCR crystal structures has provided mechanistic insights. This review will present current understanding of the key roles of ECLs in ligand binding, activation and regulation of both family A and family B GPCRs.

The pharmacology of adrenomedullin receptors and their relationship to CGRP receptors.

Adrenomedullin (AM) has two specific receptors formed by the calcitonin-receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3. These are known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the CGRP1 receptor, composed of CL and RAMP1. The AM1 receptor has a high degree of selectivity for AM over CGRP and other peptides, and AM22–52 is an effective antagonist at this receptor. By contrast, the AM2 receptor shows less specificity for AM, having appreciable affinity for βCGRP. Here, CGRP8–37 is either equipotent or more effective as an antagonist than AM22–52, depending on the species from which the receptor components are derived. Thus, under the appropriate circumstances it seems that βCGRP might be able to activate both CGRP1 and AM2 receptors and AM could activate both AM1 and AM2 receptors as well as CGRP1 receptors. Current peptide antagonists are not sufficiently selective to discriminate between these three receptors. The CGRP-selectivity of RAMP1 and RAMP3 may be conferred by a putative disulfide bond from the N-terminus to the middle of the extracellular domain of these molecules. This is not present in RAMP2.

Regulation of signal transduction by calcitonin gene-related peptide receptors.

Calcitonin gene-related peptide (CGRP) plays a pivotal role in migraine, activating its cognate receptor to initiate intracellular signalling. This atypical receptor comprises a distinct assembly, made up of a G protein-coupled receptor (GPCR), a single transmembrane protein, and an additional protein that is required for Gα(s) coupling. By altering the expression of individual receptor components, it might be possible to adjust cellular sensitivity to CGRP. In recognition of the increasing clinical significance of CGRP receptors, it is timely to review the signalling pathways that might be controlled by this receptor, how the activity of the receptor itself is regulated, and our current understanding of the molecular mechanisms involved in these processes. Like many GPCRs, the CGRP receptor appears to be promiscuous, potentially coupling to several G proteins and intracellular pathways. Their precise composition is likely to be cell type-dependent, and much work is needed to ascertain their physiological significance.

The pharmacology of Adrenomedullin 2/Intermedin

Adrenomedullin 2 (AM2) or intermedin is a member of the calcitonin gene‐related peptide (CGRP)/calcitonin family of peptides and was discovered in 2004. Unlike other members of this family, no unique receptor has yet been identified for it. It is extensively distributed throughout the body. It causes hypotension when given peripherally, but when given into the CNS, it increases blood pressure and causes sympathetic activation. It also increases prolactin release, is anti‐diuretic and natriuretic and reduces food intake. Whilst its effects resemble those of AM, it is frequently more potent. Some characterization of AM2 has been done on molecularly defined receptors; the existing data suggest that it preferentially activates the AM2 receptor formed from calcitonin receptor‐like receptor and receptor activity modifying protein 3. On this complex, its potency is generally equivalent to that of AM. There is no known receptor‐activity where it is more potent than AM. In tissues and in animals it is frequently antagonised by CGRP and AM antagonists; however, situations exist in which an AM2 response is maintained even in the presence of supramaximal concentrations of these antagonists. Thus, there is a partial mismatch between the pharmacology seen in tissues and that on cloned receptors. The only AM2 antagonists are peptide fragments, and these have limited selectivity. It remains unclear as to whether novel AM2 receptors exist or whether the mismatch in pharmacology can be explained by factors such as metabolism.

International Union of Pharmacology. LXIX. Status of the Calcitonin Gene-Related Peptide Subtype 2 Receptor

Historically, calcitonin gene-related peptide (CGRP) receptors have been divided into two classes, CGRP1 and CGRP2. After the cloning of calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMPs), it became clear that the CGRP1 receptor was a complex between CLR and RAMP1. It is now apparent that the CGRP2 receptor phenotype is the result of CGRP acting at receptors for amylin and adrenomedullin. Accordingly, the term “CGRP2” receptor should no longer be used, and the “CGRP1” receptor should be known as the “CGRP” receptor.

Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines.

The calcitonin receptor‐like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene‐related peptide (CGRP) and/or adrenomedullin in transfected cells. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. We confirmed CGRP subtype 1 receptor (CGRP1) pharmacology in SK‐N‐MC neuroblastoma cells. L6 myoblast cells expressed both CGRP1 and adrenomedullin receptors whereas Rat‐2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP2 receptor pharmacology for Col‐29 colonic epithelial cells, which, instead were CGRP1‐like in this study. L6, SK‐N‐MC and Col‐29 cells expressed mRNA for RAMP1 and RAMP2 but Rat‐2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. SK‐N‐MC, Col‐29 and Rat‐2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus, circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.