Receptor activity-modifying proteins (RAMPs) are single-transmembrane proteins that transport the calcitonin receptor-like receptor (CRLR) to the cell surface. RAMP 1-transported CRLR is a calcitonin gene-related peptide (CGRP) receptor. RAMP 2- or RAMP 3-transported CRLR is an adrenomedullin receptor. The role of RAMPs beyond their interaction with CRLR, a class II G protein-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 16-amino acid insert in intracellular domain 1 (hCTR(I1-)) into COS-7 cells induced specific 125 I-labeled rat amylin binding. RAMP 2 or vector cotransfection did not cause significant increases in specific amylin binding. Competition-binding characterization of the RAMP- induced amylin receptors revealed two distinct phenotypes. The RAMP 1- derived amylin receptor demonstrated the highest affinity for salmon CT (IC 50 , 3.01 ± 1.44 x 10 -10 M), a high to moderate affinity for rat amylin (IC 50 , 7.86 ± 4.49 x 10 -9 M) and human CGRPα (IC 50 , 2.09 ± 1.63 x 10 -8 M), and a low affinity for human CT (IC 50 , 4.47 ± 0.78 x 10 -7 M). In contrast, whereas affinities for amylin and the CTs were similar for the RAMP 3-derived receptor, the efficacy of human CGRPα was markedly reduced (IC 50 , 1.12 ± 0.45 x 10 -7 M; P < .05 versus RAMP 1). Functional cyclic AMP 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 1 indicated that, when transfected alone, RAMP 1 almost exclusively was located intracellularly. Cotransfection with calcitonin receptor (CTR)(I1-) induced cell surface expression of RAMP 1. The results of experiments cross-linking 125 I-labeled amylin to RAMP 1/hCTR-transfected cells with bis succidimidyl suberate were suggestive of a cell-surface association of RAMP 1 and the receptors. Our data suggest that in the CT family of receptors, and potentially in other 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.