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]hαCGRP binding was inhibited by rαCGRP(8–37), rαCGRP and rβCGRP 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 rβCGRP and rαCGRP 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]hαCGRP binding was inhibited by rαCGRP(8–37), rβCGRP, rαCGRP, 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 rαCGRP and rβCGRP. 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.