Distinct GRK2 Recruitment Mechanisms Differentially Shape GPCR Signaling Bias

Abstract Biased GPCR agonists that selectively engage G proteins or arrestins show therapeutic promise, but their development has been challenging. Key to understanding biased signaling is GRK2, which phosphorylates many GPCRs and facilitates arrestin recruitment. Canonically, GRK2 binds GPCRs only after being recruited to the plasma membrane by Gβγ, implying that arrestin recruitment requires prior G protein activation and challenging the feasibility of developing highly biased agonists. However, emerging evidence suggests that certain GPCRs, including dopamine D2 receptor, can bind GRK2 independently of Gβγ. Here, we combined a robust GRK2 recruitment assay and functional analyses to show that Gβγ-dependent and -independent GRK2 recruitment have distinct dynamics, signaling consequences, and impacts on bias. We further demonstrate that Gβγ-independent GRK2 recruitment facilitates arrestin recruitment to diverse GPCRs, revising the canonical model of GRK2 function. These findings highlight the need to account for distinct GRK2 recruitment mechanisms in the design of biased GPCR agonists.