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.

Adrenomedullin 2/intermedin: a putative drug candidate for treatment of cardiometabolic diseases

“…Gα s is the main signal pathway activated by CRLR‐ and CTR‐based receptors (Hong et al , ), but RAMPs influence the biased activation of CRLR and CTR in a ligand‐dependent manner. In HEK‐293 cells treated with ADM2/IMD 1–47 , the CGRP receptors display a bias toward Gα s signalling; AM 1 receptors shows a bias toward Gα q signalling; and AM 2 receptors show bias toward Gα s signalling (Weston et al , ) (Figure ).…”

“…However, when HEK‐293 cells are treated with CGRP, ADM or ADM2, the G protein selectivity of CRLR is also changed. Now, CGRP receptors displays a bias toward Gα s signalling in response to CGRP and change to a Gα i bias, in response to ADM. AM 1 receptors shows a bias toward Gα s when treated with CGRP and ADM. AM 2 receptors show bias toward Gα s in response to ADM and change to Gα q in response to CGRP (Weston et al , ). Therefore, the biased activation of CRLR by different peptides is another mechanism underlying the differences in effects between ADM2, CGRP and ADM.…”

Don’t stress about CRF: assessing the translational failures of CRF1antagonists

“…Thus, perhaps small molecules that: 1) act as non-selective antagonists at both CRF subtypes, 2) exhibit inverse agonist activity, or 3) also modulate activities of other CRF system molecules, such as CRF-BP or receptor activity modifying protein-2 (RAMP2) (Wooten et al 2013; Weston et al 2016; Gingell et al 2016) would have greater therapeutic activity than the many selective CRF 1 neutral antagonists tested to date. Similarly, the structural manner of binding the receptor may be important; perhaps small molecules that bind differentially to certain residues of the atypical allosteric binding site (e.g., compare MTIP vs. CP-376395; Xu et al 2015) may slow the antagonist’s escape kinetics (Bai et al 2014) or direct its anti-signaling pathway bias (Suen et al 2014; Zhang et al 2016).…”

GLP-1R is downregulated in beta cells of NOD mice and T1D patients

“…Islets were isolated as previously described (59) and dispersed to single cell suspension using Enzyme Free Dissociation Buffer (Gibco). Islets were seeded in a 384-well Optiplate (PerkinElmer) (4000 cells per well) and stimulated with either GLP-1 or GIP for 30 minutes prior to measurement of cAMP accumulation using a LANCE® Ultra cAMP Detection Kit (PerkinElmer) in accordance with the manufacturer's instructions and as described previously (61,62). Results are presented as % of total stimulation with 10 µM forskolin (a direct activator of adenylyl cyclase).…”