1 The calcitonin receptor-like receptor (CRLR) and speci®c receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenomedullin in transfected cells. 2 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. Speci®c human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. 3 We con®rmed CGRP subtype 1 receptor (CGRP 1 ) pharmacology in SK-N-MC neuroblastoma cells. L6 myoblast cells expressed both CGRP 1 and adrenomedullin receptors whereas Rat-2 ®broblasts expressed only adrenomedullin receptors. In contrast we could not con®rm CGRP 2 receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP 1 -like in this study.

Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin, calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP(1)) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP(1) receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via G(s) and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern.

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Adrenomedullin (ADM) is a multifunctional regulatory peptide with its effects mediated by specific receptors. Here we examined the characteristics of the ratCRLR/humanRAMP3 combination stably expressed in HEK 293 cells with respect to binding of calcitonin family peptides and desensitisation of the ADM cAMP response following pre-exposure to ADM or other peptides. HEK 293 cells were stably transfected with rat CRLR and human RAMP3. Northern blot analysis of RNA extracted from these cells showed high expression of CRLR and RAMP3 and barely detectable levels of RAMPs 1 and 2. I-ratADM binding to RAMP3/CRLR was competed for by unlabelled rat ADM with an IC 50 of 9 nM. CGRP and other calcitonin family members competed with 125 I-ratADM more weakly with the following IC 50 values; α-CGRP 168 nM, β-CGRP 69 nM, CGRP 8-37 61 nM, cys(ACM2,7)CGRP 270 nM, human ADM 22-52 220 nM, rat amylin 117 nM, rat calcitonin >1000 nM. Both ADM and CGRP caused elevation of cAMP with ADM being the most potent (EC 50 1.5 ± 1.3 nM vs. 18.0 ± 1.1 nM, n = 3). A 2-h preincubation of these cells with 100 nM ADM resulted in a 93 ± 1% (n = 3) reduction in the subsequent cAMP response to 100 nM ADM. A similar effect was seen with ADM 13-50 but α-CGRP was less effective at this concentration. The desensitisation caused by ADM was not mimicked by 1 mM dibutryl cAMP nor inhibited by pretreatment with the protein kinase A inhibitor H-89 (100 to 500 nM). Thus the ratCRLR/humanRAMP3 combination forms a receptor with higher affinity for ADM than CGRP which is potently desensitised by ADM. This desensitisation was not due to the activation of PKA.