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

The calcitonin receptor-like receptor (CRLR) requires novel receptor-activi ty-modifying proteins (RAMPs) for its function as an adrenolnedullin (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 54% amino a cid sequence identity with respect to human RAMP1, -2 and -3. On Northern b lot analysis of polyA(+) RNA mouse RAMP1 (mRAMP1) encoding mRNA with an app arent size of 0.8 kb was predominantly observed in embryonic and adult brai n and lung and in adult skeletal muscle. Mouse RAMP2 encoding 0.8 and 1.2 k b mRNA were recognized in all tissues analyzed with the highest levels in e mbryonic 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 e mbryonic and adult brain. In COS-7 cells co-expressing rat CRLR (rCRLR) and mRAMP1, [I-125]h alpha CGRP binding was inhibited by r alpha CGRP(8-37), r alpha CGRP and r beta CGRP with IC50 of 1.4 +/- 0.5, 4.5 +/- 0.6 and 7 +/- 0.3 nM, respectively. CyclicAMP accumulation was maximally stimulated tenf old by r beta CGRP and r alpha CGRP with EC50 of 0.65 +/- 0.67 and 0.86 +/- 0.6 nM. In the same cells co-expressing rCRLR and mRAMP2, binding of [I-12 5]r-ADM was displaced by rADM and rADM(20-50) with IC50 of 1.9 +/- 0.5 and 3.4 +/- 1.4 nM, respectively, and a maximal sevenfold stimulation of cAMP a ccumulation was observed with rADM with an EC50 of 0.82 +/- 0.85 nM. In the cells co-expressing rCRLR and mRAMP3, [I-125]h alpha CGRP binding was inhi bited by r alpha CGRP(8-37), r beta CGRP, r alpha CGRP, rADM and rADM(20-50 ) with IC50 between 3 and 22 nM. cAMP accumulation was stimulated by rADM w ith an EC50 of 5.1 +/- 2.7 nM that was 12-fold and 11-fold lower than that of r alpha CGRP and r beta CGRP. In conclusion. mouse RAMPI, -2 and -3 exhi bit high amino acid sequence homology to the corresponding human RAMPs. Go- expression of rCRLR with mRAMP1, -2 or -3 in COS-7 cells revealed distinct CGRP-, ADM- or ADM/CGRP receptors. (C) 2000 Published by Elsevier Science I reland Ltd. All rights reserved.