Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

Abstract

The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.This work was supported by the National Heart Foundation of New Zealand (H.W.), the School of Biological Sciences, University of Auckland seed fund (H.W.), the BBSRC (G.L. - BB/M00015X/1), (D.P. - BB/M000176/1), (C.A.R. - BB/M006883/1), a BBSRC Doctoral Training Partnership (M.H. – BB/JO14540/1), an MRC Doctoral Training Partnership (I.W. - MR/J003964/1), a Warwick Impact Fund (C.W., G.L.), a Warwick Research Development Fund (C.W., G.L.) grant number (RD13301) and the Warwick Undergraduate Research Scholarship Scheme (A.S and R.H).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by the American Society for Biochemistry and Molecular Biology