Mouse receptor-activity-modifying proteins 1, -2 and -3: amino acid sequence, expression and function.

Abstract

The calcitonin receptor-like receptor (CRLR) requires novel receptor-activity-modifying proteins (RAMPs) for its function as an adrenomedullin (ADM) or a calcitonin (CT) gene-related peptide (CGRP) receptor. Here, mouse cDNA clones representing expressed sequence tags (ESTs) in the GenEMBL database have been identified. They encode for proteins with 70, 68 and 84% amino acid sequence identity with respect to human RAMP1, -2 and -3. On Northern blot analysis of polyA(+) RNA mouse RAMP1 (mRAMP1) encoding mRNA with an apparent size of 0.8 kb was predominantly observed in embryonic and adult brain and lung and in adult skeletal muscle. Mouse RAMP2 encoding 0.8 and 1.2 kb mRNA were recognized in all tissues analyzed with the highest levels in embryonic brain, lung and gut and in adult heart, lung, skeletal muscle and brain. A single 1.2 kb mRAMP3 encoding transcript was mainly expressed in embryonic and adult brain. In COS-7 cells co-expressing rat CRLR (rCRLR) and mRAMP1, [125I]halphaCGRP binding was inhibited by ralphaCGRP(8-37), ralphaCGRP and rbetaCGRP with IC(50) of 1.4+/-0.5, 4.5+/-0.6 and 7+/-0.3 nM, respectively. CyclicAMP accumulation was maximally stimulated tenfold by rbetaCGRP and ralphaCGRP with EC(50) of 0. 65+/-0.67 and 0.86+/-0.6 nM. In the same cells co-expressing rCRLR and mRAMP2, binding of [125I]rADM was displaced by rADM and rADM(20-50) with IC(50) of 1.9+/-0.5 and 3.4+/-1.4 nM, respectively, and a maximal sevenfold stimulation of cAMP accumulation was observed with rADM with an EC(50) of 0.82+/-0.85 nM. In the cells co-expressing rCRLR and mRAMP3, [125I]halphaCGRP binding was inhibited by ralphaCGRP(8-37), rbetaCGRP, ralphaCGRP, rADM and rADM(20-50) with IC(50) between 4 and 22 nM. cAMP accumulation was stimulated by rADM with an EC(50) of 5.1+/-2.7 nM that was 12-fold and 11-fold lower than that of ralphaCGRP and rbetaCGRP. In conclusion, mouse RAMP1, -2 and -3 exhibit high amino acid sequence homology to the corresponding human RAMPs. Co-expression of rCRLR with mRAMP1, -2 or -3 in COS-7 cells revealed distinct CGRP-, ADM- or ADM/CGRP receptors.