GPCR Kinase Knockout Cells Reveal the Impact of Individual GRKs on Arrestin Binding and GPCR Regulation.

GPCR kinases (GRKs) regulate GPCR interactions and thus functions. Here, the authors report a comprehensive panel of GRK knockout cells, used to assess the GRK-specific beta-arrestin recruitment. Selective engagement of GRKs induces distinct GPCR-beta-arrestin complexes. G protein-coupled receptors (GPCRs) activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and the formation of receptor-arrestin complexes. However, the impact of individual GRKs on arrestin binding is not clear. We report the creation of eleven combinatorial HEK293 knockout cell clones lacking GRK2/3/5/6, including single, double, triple and the quadruple GRK knockout. Analysis of beta-arrestin1/2 interactions for twelve GPCRs in our GRK knockout cells enables the differentiation of two main receptor subsets: GRK2/3-regulated and GRK2/3/5/6-regulated receptors. Furthermore, we identify GPCRs that interact with beta-arrestins via the overexpression of specific GRKs even in the absence of agonists. Finally, using GRK knockout cells, PKC inhibitors and beta-arrestin mutants, we present evidence for differential receptor-beta-arrestin1/2 complex configurations mediated by selective engagement of kinases. We anticipate our GRK knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and beta-arrestin complex formation.