title: Mapping the putative G protein-coupled receptor (GPCR) docking site on GPCR kinase 2: insights from intact cell phosphorylation and recruitment assays.
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.
authors: From the Department of Biochemistry and the Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors: Alexandre
authors: A
authors: Beautrait
authors: the Departments of Chemistry and Biochemistry and.
authors: Kevin R
authors: KR
authors: Michalski
authors: the Departments of Chemistry and Biochemistry and.
authors: Thomas S
authors: TS
authors: Lopez
authors: the Departments of Chemistry and Biochemistry and.
authors: Katelynn M
authors: KM
authors: Mannix
authors: the Departments of Chemistry and Biochemistry and.
authors: Devin J
authors: DJ
authors: McDonald
authors: the Departments of Chemistry and Biochemistry and.
authors: Amber R
authors: AR
authors: Cutter
authors: the Departments of Chemistry and Biochemistry and.
authors: Christopher B
authors: CB
authors: Medina
authors: the Departments of Chemistry and Biochemistry and.
authors: Aaron M
authors: AM
authors: Hebert
authors: Biology, Siena College, Loudonville, New York 12211, and.
authors: Charnelle J
authors: CJ
authors: Francis
authors: From the Department of Biochemistry and the Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors: Michel
authors: M
authors: Bouvier
authors: the Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109.
authors: John J G
authors: JJ
authors: Tesmer
authors: Biology, Siena College, Loudonville, New York 12211, and sternemarr@siena.edu.
authors: Rachel
authors: R
authors: Sterne-Marr
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2
keywords: Adrenergic Receptor; Bioluminescence Resonance Energy Transfer (BRET); G Protein-coupled Receptor (GPCR); GPCR Kinase (GRK); Intact Cell Phosphorylation; Mutagenesis; Phosphorylation