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.