G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5) Exhibit Selective Phosphorylation of the Neurotensin Receptor in Vitro

G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activ...