Role of the GRK2/3 N-terminus in discriminating the endocytic effects of opioid agonist drugs.

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Endocytosis of the μ-type opioid receptor (MOR) is a fundamentally important cellular regulatory process that is characteristically driven less effectively by partial relative to full agonist ligands. Such agonist-selective endocytic discrimination depends on how strongly drugs promote MOR binding to β-arrestins and this, in turn, depends on how strongly they stimulate phosphorylation of the MOR cytoplasmic tail by GPCR kinases (GRKs) from the GRK2/3 subfamily. While these relatively 'downstream' steps in the agonist-selective endocytic pathway are now well defined, it remains unclear how agonist-bound receptors are distinguished 'upstream' by GRKs. Focusing on GRK2 as a prototype, we show that this single GRK subtype can distinguish the endocytic activities of different MOR agonists in cells lacking other GRKs, and that agonist-selectivity is introduced at the most upstream step of GRK2 binding to MOR. This interaction requires prior membrane recruitment of GRK2 by its conserved PH domain and is enhanced by phosphorylation of the MOR tail, but neither reaction can explain the high degree of agonist-selectivity in the observed interaction of GRK2 with MOR. We identify the N-terminal domain (NTD) of GRK2, which is identical in GRK3, as a discrete element required for the full agonist-selectivity of MOR-GRK2 interaction and show that the NTD is also required for GRK2 to promote MOR endocytosis after it is bound. We propose a simple cellular mechanism of upstream agonist discrimination that is organized as a series of biochemical checkpoints and utilizes the NTD as an agonist-selective sensor. Significance Statement This study investigates how GPCR kinases (GRKs) distinguish the effects of opioid agonist drugs on regulated endocytosis of the μ-opioid receptor (MOR). It shows that a single GRK subtype is sufficient to determine the agonist-selectivity of MOR internalization, agonists are distinguished by how strongly they promote GRK2 recruitment by MOR, and the GRK2/3 N-terminus is a key determinant of agonist discrimination.