CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells

Abstract

Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can “re-route” the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling.

The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activation

“…All pharmacological data was analyzed in GraphPad Prism v9.0 (GraphPad Software, San Diego). Data were fitted to obtain concentration-response curves using either the three-parameter logistic equation to obtain values of E max and pEC 50 or the operational model of agonism (44) as described previously (33,34), to obtain efficacy (t) and equilibrium disassociation constant (K A ) values. Statistical differences were analysed using one-way ANOVA followed by Dunnett's post-hoc (for comparisons amongst more than two groups).…”

“…The means of individual experiments were combined to generate the concentration-response curves displayed in the figures. Having obtain values for t and K A these were then used to quantify signaling bias as the change in Log(t/K A ) relative to WT and a reference signaling pathway (ERK1/2) as described previously (34).…”

“…Cells were stimulated with ligand for 5 min, before lysis using the supplied lysis buffer and assayed for ERK1/2 phosphorylation using the phospho-ERK (Thr202/Tyr204) Cellular Assay Kit (Cisbio). Plates were read using a Mithras LB 940 multimode microplate reader and data normalized to the maximal response to 100 mM phorbol 12-myristate 13-acetate (PMA, Sigma) (34).…”

“…Here we study the role of ICL1-H8 unit in the activation of class B1 GPCRs and in particular, the significance of a motif previously referred to as the [K/R]KLH motif (32) within ICL1 which mediates its interactions with H8. We have primarily concentrated on the calcitonin receptor-like receptor (CLR), a receptor we have studied (33,34), in association with receptor activity-modifying protein 1 (RAMP1), this forms a receptor for CGRP which can also recognize the related peptide adrenomedullin (AM) (35). We have also examined the glucagon receptor (GCGR) and two splice variants of the CRFR1.…”

The Vascular-Dependent and -Independent Actions of Calcitonin Gene-Related Peptide in Cardiovascular Disease

“…The current consensus is that, upon neuronal depolarization, αor β-CGRP is released from sensory neurons via calciumdependent exocytosis to bind to its receptor situated on the plasma membrane of several cell types including, but not limited to, smooth muscle cells (Argunhan et al, 2021), endothelial cells (Gray and Marshall, 1992a) and cardiomyocytes (Clark et al, 2021). The receptor complex is composed of a seven domain G-protein coupled receptor (GPCR) known as calcitonin receptor-like receptor (CRLR) and a single transmembrane protein recognized as receptor activity modifying protein-1 (RAMP1).…”