Three receptor-activity-modifying proteins define calcitonin gene-related peptide or adrenomedullin selectivity of the mouse calcitonin-like receptor in COS-7 cells

Receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are heterodimeric complexes of the calcitonin-like receptor (CLR) together with associated receptor-activity-modifying proteins (RAMP)1, -2 or -3. The RAMP define the specificity of the CLR for CGRP or AM. Here, mouse (m)CLR/mRAMP1, -2 and -3 were expressed in COS-7 cells that lack detectable CGRP and AM receptors. myc epitope-tagged non-glycosylated mRAMP1 required V5-tagged mCLR for its translocation to the cell surface. The glycosylated myc-mRAMP2 and -3, on the other hand, were expressed at the cell surface in the absence of co-transfected mCLR. Selective binding of 125I]h alpha CGRP to mCLR/mRAMP1 expressing cells was inhibited by rat (r)alpha CGRP(1-37) and the CGRP antagonist r alpha CGRP(8-37) with IC(50) of 7.0+/-1.6 nM and 1.0+/-0.1 nM (mean+/-SEM). rAM(1-50) and the AM antagonist rAM(20-50) inhibited 125I]h alpha CGRP binding at over 36-fold higher concentrations than r alpha CGRP. In mCLR/mRAMP2 expressing cells, selective 125I]rAM binding was inhibited by rAM(1-50) and -(20-50) with IC(50) of 8.9+/-2.6 nM and 34+/-9 nM. r alpha CGRP(1-37) and -(8-37) displaced the binding at over 25-fold higher concentrations. mCLR/mRAMP3 expressing cells recognized both 125I]h alpha CGRP and -rAM. The IC(50) of rAM and r alpha CGRP(8-37) ranged between 5.8 and 7.0 nM, and those of r alpha CGRP and rAM(20-50) were only 4- to 8-fold higher. r alpha CGRP and rAM stimulated and r alpha CGRP(8-37) and rAM(20-50) antagonized mCLR/mRAMP1, -2 and -3 mediated cAMP formation with relative potencies that reflected the observed CGRP and AM selectivity of the three receptor types. In conclusion, mCLR/mRAMP1 and -2 are CGRP- and AM-selective receptors, respectively, whereas mCLR/mRAMP3 is an AM/CGRP receptor.