Matthees, E. S. | bioRxiv Science

bioRxiv2024

Relating GPCR domains with functionality: receptor helix-bundle and C-terminus differentially influence GRK-specific functions and β-arrestin-mediated regulation

G protein-coupled receptors (GPCRs) orchestrate diverse physiological responses via intracellular signaling through G proteins, GPCR kinases (GRKs), and arrestins. While the role of G proteins in receptor signaling is well-established, the contributions of GRKs and arrestins remain incompletely understood. Here, we investigate the influence of arrestin-interacting GPCR domains (helix-bundle/C-terminus) on {beta}-arrestin conformations and functions using refined biosensors and advanced cellular knockout systems. By focusing on prototypical class A (b2AR) and B (V2R) receptors and their chimeras (b2V2/V2b2), we can now characterize differential {beta}-arrestin conformational changes as primarily mediated by the receptor C-terminus or helix-bundle. Moreover, we demonstrate that some {beta}-arrestin-supported processes are governed by distinct receptor domains, such as ERK1/2 activation (helix-bundle) and arrestin co-internalization (C-terminus), while others depend on the overall GPCR configuration, such as receptor internalization. Our findings elucidate how individual GPCR domains dictate downstream signaling events, shedding light on the structural basis of receptor-specific signaling and regulation.

pharmacology and toxicology↗

bioRxiv2023

GRK specificity and beta gamma dependency determines a GPCR`s potential in biased agonism

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent {beta}-arrestin recruitment. Recently, it was shown that GPCRs differentially depend on GRK2/3, GRK2/3/5/6 or GRK5/6 for their regulation. The four ubiquitously expressed GRKs are classified into the cytosolic GRK2/3 and the membrane-tethered GRK5/6 subfamily. In vitro studies revealed that GRK2/3 interact with the membrane-localized G protein {beta}{gamma}-subunits. Yet, the role of this interaction as crosslink between G protein activation and {beta}-arrestin binding to GPCRs remained strongly underappreciated. Here we systematically show that the G{beta}{gamma}-GRK2/3 interaction is key for these GRKs to mediate {beta}-arrestin2 binding to Gs-, Gi- and Gq-coupled GPCRs. In our GRK2/3/5/6 knockout cells, without endogenous GRK background, the utilized GRK2/3 mutants devoid of the G{beta}{gamma} interaction site significantly diminished {beta}-arrestin2 recruitment to the beta-2 adrenergic receptor (b2AR), muscarinic M2 and M5 acetylcholine receptors (M2R, M5R). This effect was overwritten by artificially tethering GRK2/3 via a CAAX motif to the plasma membrane independently of free G{beta}{gamma}. Hence, the membrane recruitment is crucial for GRK2/3-mediated {beta}-arrestin2 binding to GPCRs, which is naturally induced via the G{beta}{gamma} interaction. This connects the {beta}-arrestin interaction for GRK2/3-regulated receptors inseparably with the associated G protein activation. We outline a theoretical framework of how GRK dependence on free G{beta}{gamma} can determine a GPCRs potential in biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we propose that it will likely be mechanistically unattainable to create {beta}-arrestin-biased ligands for the subgroup of GRK2/3-regulated GPCRs, while GRK5/6-mediated receptor regulation is independent from G{beta}{gamma} availability. Accordingly, one should first determine the GRK specificity of a GPCR to ultimately assess the receptors potential for the development of biased ligands.

pharmacology and toxicology↗