Adrenomedullin (ADM) and alpha- and beta-calcitonin (CT) gene-related peptide (alpha-, betaCGRP) are structurally related vasodilatory peptides with homology to CT and amylin. An originally orphan human CT receptor-like receptor (hCRLR) is a Gs protein-coupled CGRP or ADM receptor when coexpressed with recently identified human single transmembrane domain receptor activity modifying proteins 1 (hRAMP1) or -2 (hRAMP2), respectively. Here, the function of the rat CRLR homologue (rCRLR) has been investigated in rat osteoblast-like UMR-106 cells and in COS-7 cells, in the absence and presence of hRAMP1 and -2 and combinations thereof. Transient expression of rCRLR in UMR-106 cells revealed an ADM receptor, and [125I]rat (r) ADM binding was enhanced with hRAMP2 and inhibited by 50% when hRAMP1 was coexpressed. Detectable [125I]h alphaCGRP binding required the presence of hRAMP1, and the expression of CGRP binding sites was unaffected by coexpressed hRAMP2. Specificity of ADM binding sites in [125I]rADM binding inhibition experiments was reflected by an over 1000-fold higher potency of rADM [half-maximal effective concentration = 0.19 +/- 0.05 nM (mean +/- SEM, n = 4)], compared with r alphaCGRP and r betaGRP, to induce a cAMP-responsive luciferase reporting gene (CRE-luc). In rCRLR and hRAMP1 cotransfected cells, expressing predominantly CGRP binding sites, r betaCGRP, r alphaCGRP, and rADM induced CRE-luc with half-maximal effective concentration of 0.27 +/- 0.17 nM, 0.37 +/- 0.27 nM, and 1.4 +/- 0.9 nM, respectively. In COS-7 cells, the results were comparable, but rCRLR required coexpressed hRAMP2 for ADM receptor function. This is consistent with higher levels of endogenous RAMP2 encoding messenger RNA in UMR-106, compared with COS-7 cells. In conclusion, the recognition of RAMP1 and -2 as mediators of CRLR expression as a CGRP or ADM receptor has been extended, with evidence that endogenous RAMP2 is sufficient to reveal an ADM receptor in UMR-106 cells. Inhibition of RAMP2-evoked ADM receptor expression by RAMP1 and generation of a CGRP receptor is consistent with competitive interactions of the different RAMPs with rCRLR.