Mapping the Putative G Protein-Coupled Receptor (GPCR) Docking Site on GPCR Kinase 2: Insights from Intact Cell Phosphorylation and Recruitment Assays.

The Journal of biological chemistry

PubMedID: 25049229

Beautrait A, Michalski KR, Lopez TS, Mannix KM, McDonald DJ, Cutter AR, Medina CB, Hebert AM, Francis CJ, Bouvier M, Tesmer JJ, Sterne-Marr R. Mapping the Putative G Protein-Coupled Receptor (GPCR) Docking Site on GPCR Kinase 2: Insights from Intact Cell Phosphorylation and Recruitment Assays. J Biol Chem. 2014;.
G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and ß-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (aN), the small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the aN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation, in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer (BRET)-based assay to monitor the recruitment of GRK2 to activated a2A-adrenergic receptors (a2A-ARs) in living cells. The BRET signal exhibited a biphasic response to norepinephrine concentration suggesting that GRK2 is recruited to Gß? and a2A-AR with EC50s of 15 nM and 8 µM, respectively. We show that mutations in aN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2 including Leu(4), Val(7), Leu(8), Val(11), and Ser(12), directly interacts with receptors, whereas residues such as Asp(10), Tyr(13), Ala(16), Met(17), Gly(475), Val(477), and Ile(485) are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggests that all three GRK subfamilies make conserved interactions with GPCRs, but there may be unique interactions that influence selectivity.