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.
Highlights
Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor
Using human endothelial cells which endogenously express the adrenomedullin 1 (AM1) receptor, we demonstrate that biased agonism is present and has a fundamental role in the function of peptide hormones acting on primary human cells
While there are many reports of biased agonism for G protein-coupled receptors (GPCRs) in recombinant systems (e.g.19–21), few examples have been documented in primary human cells
Summary
Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. 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). When co-expressed with one of three receptor-activity modifying proteins, (RAMPs), it can be activated by distinct endogenous agonists; calcitonin-gene related polypeptide (CGRP), adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2) This makes it a good system to investigate the role of bias for endogenous ligands. Beyond the Gα subunits CLR has been reported to couple to β-arrestins[13] inducing internalisation, it has been suggested that this interaction can lead to its own signalling events possibly promoting cell proliferation[14] Despite this high potential for agonist-induced pleiotropy, CLR remains most closely associated with adenylyl cyclase activation and generation of cAMP. We chose to focus on RAMP1 and RAMP2 as the CGRP and AM1 receptors are the best described
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