Characterization of the Human Calcitonin Gene-Related Peptide Receptor Subtypes Associated with Receptor Activity-Modifying Proteins

Coexpression of receptor activity-modifying proteins (RAMPs) with calcitonin receptor 2 (CTR2) or calcitonin receptor-like receptor (CRLR) leads to the formation of four functional heterodimeric receptors for human calcitonin gene-related peptide (hCGRP). In this study, we transfected hCGRP receptors into human embryonic kidney 293 cells and examined their pharmacological profiles using three dominant-negative (DN) RAMP mutants and various hCGRPα analogs. Fluorescence-activated cell-sorting analysis revealed that their cotransfection with CTR2 induced cell surface expression of all three RAMPs, and the three CTR2/RAMP heterodimers mediated equivalent levels of cAMP production in response to hCGRPα that were approximately 50-fold greater than were seen with CTR2 alone. By contrast, [Tyr⁰]hCGRPα binding and signaling were markedly weaker with CTR2/RAMP2 or -3 than with CTR2/RAMP1 or CRLR/RAMP1; likewise, ¹²⁵I-[His¹⁰]hCGRPα bound most potently to CTR2/RAMP1. When CTR2 was coexpressed with DN RAMP1 or -2, hCGRPα-evoked responses were similar to those seen with CTR2 alone, despite the expression of both CTR2 and DN RAMP at the cell surface. But coexpression of DN RAMP3 with CTR2 significantly diminished hCGRPα signaling compared with that seen with CTR2 alone, indicating that DN RAMP3 is able to function as a negative regulator of CTR2 function. Competition experiments showed the relative agonist sensitivity of the four receptors to be hCGRPα > [Tyr⁰]hCGRPα > [Cys(Et)^2,7]hCGRPα > [Cys(ACM)^2,7]hCGRPα. Of the linear analogs, [Cys(ACM)^2,7]hCGRPα (ACM, acetylmethoxy) enhanced cAMP formation only via CTR2/RAMP1, whereas [Cys(Et^2,7)]hCGRPα acted via CRLR/RAMP1 and somewhat less potently via CTR2/RAMP1. Thus, among the three CGRP_8–37-insensitive receptors, CTR2/RAMP1 is most sensitive to the two linear analogs, suggesting that it could be classified as a CGRP2 receptor. Moreover, the combined use of iodinated CGRPα analogs may be useful for defining the CGRP1 receptor.