Pharmacological characterization of receptor-activity-modifying proteins (RAMPs) and the human calcitonin receptor

Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMPS, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMPS coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMPS and 9.45 +/- 0.07 with RAMPS. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMPS and 8.2 +/- 0.35 with RAMPS. The finding suggests that RAMPS might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems. (C) 2000 Elsevier Science Inc. All rights reserved.