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

Alexandre Beautrait,Kevin R Michalski,Thomas S Lopez,Katelynn M Mannix,Devin J Mcdonald,Amber R Cutter,Christopher B Medina,Aaron M Hebert,Charnelle J Francis,Michel Bouvier,John J.G Tesmer,Rachel Sterne-Marr

doi:10.1074/jbc.m114.593178

Abstract

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase 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 αN 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-based assay to monitor the recruitment of GRK2 to activated α(2A)-adrenergic receptors (α(2A)ARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α(2A)AR with EC50 values of 15 nM and 8 μM, respectively. We show that mutations in αN (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 suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.

Highlights

Activation of GRK2 requires interaction with agonist-occupied G protein-coupled receptors (GPCRs)
The two major goals of this work were 1) to assess the roles of specific GRK2 ␣N and active site tether (AST) residues in forming a potential receptor-docking site and 2) to determine how individual residues contribute to receptor interaction, allosteric activation, or both
We created a homology model of activated GRK2 based on the GRK6-sangivamycin structure (Fig. 1)

Summary

Introduction

Activation of GRK2 requires interaction with agonist-occupied GPCRs. Results: Residues on the GRK2 N terminus and kinase domain extension collaborate to create a GPCR docking site. Significance: Mapping the GRK-GPCR interface is required to understand the mechanism and specificity of GRK activation, and, the regulation of GPCRs. G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and ␤-arrestin-dependent signaling. Expanded mutagenesis of the ␣N 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 developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated ␣2A-adrenergic receptors (␣2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to G␤␥ and ␣2AAR with EC50 values of 15 nM and 8 ␮M, respectively. We suggest that a surface of GRK2, including Leu, Val, Leu, Val, and Ser, directly interacts with receptors, whereas residues such as Asp, Tyr, Pharmacology. 3 To whom correspondence should be addressed: Biology Department, Siena

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Conclusion