Premium
Mapping G Protein‐coupled Receptor (GPCR) Docking Site on GPCR Kinase 2: New Insights from Intact Cell Studies
Author(s) -
SterneMarr Rachel,
Michalski Kevin,
Beautrait Alexandre,
Lopez Thomas,
Mannix Katelynn,
McDonald Devin,
Cutter Amber,
Francis Charnelle,
Medina Christopher,
Hebert Aaron,
Bouvier Michel,
Tesmer John
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.893.2
Subject(s) - g protein coupled receptor , g protein coupled receptor kinase , beta adrenergic receptor kinase , protein kinase domain , receptor , kinase , microbiology and biotechnology , allosteric regulation , phosphorylation , agonist , chemistry , biology , biochemistry , gene , mutant
G protein‐coupled receptor (GPCR) kinases (GRKs) desensitize GPCRs by phosphorylating agonist‐occupied receptors. GRK kinase activity is stimulated by interaction with activated receptors. The GRK 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 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 α 2A ‐adrenergic receptors (α 2A ‐ARs) in living cells. The BRET assay allowed us to distinguish GRK2 recruitment to Gβγ versus α 2A ‐AR. We suggest that one surface of GRK2 αN directly interacts with receptors, whereas the opposite face and three AST residues 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. Funding: NSF (RSM), NIH (JT), Canadian Institute for Health Research (MB), and FRSQ & Groupe de Recherche Universitaire sur le Médicament (AB).