Elsevier

Peptides

Volume 27, Issue 6, June 2006, Pages 1367-1375
Peptides

Pharmacology of the human CGRP1 receptor in Cos 7 cells

https://doi.org/10.1016/j.peptides.2005.11.014Get rights and content

Abstract

Only limited pharmacological characterization of the CGRP1 receptor, a heterodimer of the calcitonin (CT) receptor-like receptor (CL) and receptor activity-modifying protein 1 has been performed in cells that do not endogenously express RAMP2. We characterized the receptor in RAMP-deficient Cos 7 cells by measuring cAMP responses following agonist treatment in the absence or presence of antagonists. Potent cAMP responses to human α-and β-CGRP (Cys(Et)2,7)hαCGRP and human adrenomedullin (AM) were observed. Adrenomedullin15–52 was also an effective agonist of the CGRP1 receptor but human and salmon calcitonin and rat amylin were only weak agonists. As expected, BIBN4096BS and CGRP8–37 were effective antagonists of the CGRP1 receptor. (Cys(Acm)2,7)hαCGRP also antagonized CGRP responses. Antagonists of related receptors were only weakly able to inhibit CGRP responses.

Introduction

Calcitonin (CT) gene-related peptide (CGRP) is a potent vasodilator with pleiotropic pharmacological actions [3]. It belongs to the CT peptide family. This group of peptides share many structural features and functional activities. There has been a resurgence of interest in the properties of CGRP in recent years with two important discoveries; CGRP receptors were cloned [20] and the first high affinity, selective CGRP receptor antagonist was identified [9]. This antagonist, BIBN4096BS, is currently in late-stage development as a novel class of treatment for migraine and thus information regarding its mode and selectivity of action is of considerable interest [24].

Pharmacologically, two major phenotypes of CGRP receptor have been reported; CGRP1 and CGRP2 receptors [17]. The most consistent difference between them is their sensitivity (CGRP1) or insensitivity (CGRP2) to antagonism by the CGRP fragment CGRP8–37 but it has also been reported that they can be selectively activated by the linear CGRP analogs (Cys(Acm)2,7)hαCGRP and (Cys(Et)2,7)hαCGRP [17]. There is strong evidence to suggest that CGRP1 receptors are composed of heterodimeric complexes of the family B CT receptor-like receptor (CL) and the single transmembrane receptor activity modifying protein 1 (RAMP1) [26]. In contrast, the molecular nature of CGRP2 receptors has not been defined but there is evidence that AMY1(a) or AM2 receptors might contribute to reports of CGRP2 receptors; both receptors can be activated by CGRP and its linear analogs but are only weakly antagonized by CGRP8–37 [12], [15], [19]. In order to understand the contribution of different CGRP receptor subtypes to peptide physiology it is imperative to have undertaken thorough pharmacological characterization of CGRP receptors so that the in vivo use of agonists and antagonists can be appropriately interpreted.

Although the pharmacology of CGRP1 receptors is relatively well defined, there has been no detailed functional analysis of recombinant CGRP1 receptors, composed of human CL and human RAMP1. In particular, many studies of transfected CGRP1 receptors have been performed in HEK293 cells which often endogenously express RAMP2 [1]. Thus, it is unclear how much of the apparent CGRP1 receptor response to adrenomedullin (AM) is due to inherent activity at CL/RAMP1 complexes or because of the formation of AM receptors, in addition to CGRP receptors. There is a similar issue for cell lines that endogenously express CGRP1 receptor components as they also invariably express RAMP2 (e.g. SK-N-MC, L6) [5]. Furthermore, the functional effects of antagonists have not been studied in depth at CGRP1 receptors. Therefore, in this study we sought to determine agonist potencies and antagonist pA2 or pKB (affinity) values for a selection of agonists and antagonists of CT peptide family receptors at CGRP1 receptors expressed in Cos 7 cells, cells which, in our hands, do not express significant levels of endogenous RAMPs.

Section snippets

Materials

All peptides were purchased from Bachem (Bubendorf, Switzerland) except rat amylin (AMY) which was from Auspep (Parkville, Australia) and the 15–52 fragment of AM (AM15–52) was kindly provided by Professor David Coy (Tulane University Medical School, New Orleans). BIBN4096BS was a kind gift from Henri Doods (Boehringer Ingelheim). Isobutylmethylxanthine (IBMX), protein kinase A and activated charcoal were from Sigma. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were

cAMP

Mock transfection of Cos 7 cells with empty vector (pcDNA3.1) did not yield significant elevation of cAMP above basal following human CT, CGRP or AM treatment (100 nM each) (Fig. 1A). Furthermore, transfection of RAMP1 alone did not induce cAMP responses to CGRP, suggesting that endogenous CL or CTR was not present. Likewise, transfection of CL alone did not lead to elevation of cAMP levels in response to CGRP or AM indicating that there were insufficient levels of endogenous RAMPs to produce

Discussion

This study provides the first comprehensive pharmacological analysis of recombinant CGRP1 receptors expressed in cells that do not endogenously express significant levels of RAMP2. Such data provides a useful baseline for understanding the pharmacology of CGRP receptors in more complex systems. As binding studies have not historically been shown to be useful in defining CGRP receptor subtypes, we elected to functionally analyze the CGRP1 receptor in this study [7], [8], [27], [28], [31].

Acknowledgements

We would like to thank Professor David Coy (Tulane University Medical School, New Orleans) for the synthesis and provision of AM15–52. This work was supported by grants from the Auckland Medical Research Foundation, New Zealand Lottery Health fund and University of Auckland Staff Research fund. We thank Dr. David R. Poyner for critical reading of this manuscript.

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