Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
The receptor activity-modifying proteins (RAMPs) and the calcitonin receptor-like receptor (CRLR) are both required to generate adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors. A mature, fully glycosylated, form of CRLR was associated with (125)I-CGRP binding, upon co-expression of RAMP1 and CRLR. In contrast, RAMP2 and -3 promoted the expression of smaller, core-glycosylated, CRLR forms, which were linked to AM receptor pharmacology. Since core glycosylation is classically a trademark of immature proteins, we tested the hypothesis that the core-glycosylated CRLR forms the AM receptor. Although significant amounts of core-glycosylated CRLR were produced upon co-expression with RAMP2 or -3, cross-linking experiments revealed that (125)I-AM only bound to the fully glycosylated forms. Similarly, (125)I-CGRP selectively recognized the mature CRLR species upon co-expression with RAMP1, indicating that the glycosylation does not determine ligand-binding selectivity. Our results also show that the three RAMPs lie close to the peptide binding pocket within the CRLR-RAMP heterodimers, since (125)I-AM and (125)I-CGRP were incorporated in RAMP2, -3, and -1, respectively. Cross-linking also stabilized the peptide-CRLR-RAMP ternary complexes, with the expected ligand selectivity, indicating that the fully processed heterodimers represent the functional receptors. Overall, the data indicate that direct protein-protein interactions dictate the pharmacological properties of the CRLR-RAMP complexes.