Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product

Receptor activity-modifying proteins (RAMPs) are single-transmembrane prote ins that transport the calcitonin receptor-like receptor (CRLR) to the cell surface. RAMP I-transported CRLR is a calcitonin gene-related peptide (CGR P) receptor. RAMP 2- or RAMP 3-transported CRLR is an adrenomedullin recept or. The role of RAMPs beyond their interaction with CRLR, a class II G prot ein-coupled receptor, is unclear. In this study, we have examined the role of RAMPs in generating amylin receptor phenotypes from the calcitonin (CT) receptor gene product. Cotransfection of RAMP 1 or RAMP 3 with the human CT receptor lacking the 1 g-amino acid insert in intracellular domain 1 (hCTR (I1-)) into COS-7 cells induced specific I-125-labeled rat amylin binding. RAMP 2 or vector cotransfection did not cause significant increases in spec ific amylin binding. Competition-binding characterization of the RAMP-induc ed amylin receptors revealed two distinct phenotypes. The RAMP 1-derived am ylin receptor demonstrated the highest affinity for salmon CT (IC50, 3.01 /- 1.44 x 10(-10) M), a high to moderate affinity for rat amylin (IC50, 7.8 6 +/- 4.49 x 10(-9) M) and human CGRP alpha (IC50, 2.09 +/- 1.63 x 10(-8) M ), and a low affinity for human CT (IC50, 4.47 +/- 0.78 x 10(-7) M). In con trast, whereas affinities for amylin and the CTs were similar for the RAMP 3-derived receptor, the efficacy of human CGRP alpha was markedly reduced ( IC50, 1.12 +/- 0.45 x 10(-7) M; P < .05 versus RAMP 1). Functional cyclic A MP responses in COS-7 cells cotransfected with individual RAMPs and hCTR(I1 -) were reflective of the phenotypes seen in competition for amylin binding . Confocal microscopic localization of c-myc-tagged RAMP I indicated that, when transfected alone, RAMP 1 almost exclusively was located intracellular ly. Cotransfection with calcitonin receptor (CTR)(I1-) induced cell surface expression of RAMP 1. The results of experiments cross-linking I-125-label ed amylin to RAMP 1/hCTR-transfected cells with bis succidimidyl suberate w ere suggestive of a cell-surface association of RAMP 1 and the receptors. O ur data suggest that in the CT family of receptors, and potentially in othe r class II G protein-coupled receptors, the cellular phenotype is likely to be dynamic in regard to the level and combination of both the receptor and the RAMP proteins.