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 aGαs-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 RAMPdependent 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.
Bibliographical note© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Creative Commons CC-BY license.
Funding: National Heart Foundation of New Zealand; University of Auckland seed fund; BBSRC (BB/M00015X/1, BB/M000176/1 and BB/M006883/1 and BBSRC Doctoral Training Partnership Grant BB/JO14540/1); MRC Doctoral Training Partnership MR/J003964/1; Warwick Impact Fund (RD13301) from the Warwick Research Development Fund and the Warwick Undergraduate Research Scholarship Scheme.
This article contains supplemental Movies 1 and 2.
- G protein-coupled receptor
- molecular dynamics
- molecular modeling
- signal transduction
- adrenomedullin 2
- signal bias