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GPCR Kinase 2 (GRK2) Phosphorylation of the α 2A ‐Adrenergic Receptor in Intact Cells
Author(s) -
SterneMarr Rachel,
Barnett Jessica,
Hebert AaronMichael,
Weldin Erica,
Farina Zachary,
Guirguis Jeska,
Varney Mathew
Publication year - 2021
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.2021.35.s1.02094
Subject(s) - phosphorylation , beta adrenergic receptor kinase , g protein coupled receptor , g protein coupled receptor kinase , chemistry , kinase , microbiology and biotechnology , biochemistry , receptor , agonist , biology
The α 2A ‐adrenergic receptor (α 2A AR) is activated by the catecholamine norepinephrine and plays a many roles including neuromodulation, vasoconstriction in the cardiovascular system, regulation of insulin secretion and platelet aggregation. Platelet α 2A AR serves as an excellent substrate for agonist‐dependent phosphorylation by G protein‐coupled receptor (GPCR) kinase 2 (GRK2) in vitro. α 2A AR is structurally distinct from the well‐characterized beta2‐adrenergic receptor (β 2 AR) in possessing a large (~100 amino acid) third intracellular loop containing four successive serine residues that serve as phosphor‐acceptors. When overexpressed with GRK2, the receptor is phosphorylated at all 4 phospho‐sites. Our long‐term goal is to understand how GRK2 interacts with and is activated by GPCRs. We have mapped residues on GRK2 that are required for phosphorylation of the β 2 AR and recruitment to α 2A AR in intact cells. We wish to assay phosphorylation of α 2A AR in intact cells and because no commercially available α 2A AR phosphorylation site antibodies currently exist, we set out to develop such a reagent. An antibody that detects α 2A AR phosphorylation at serines 296‐299 was generated and characterized. This antibody detects an epinephrine‐stimulated signal that is increased two‐fold by transfection of GRK2 in COS‐7 cells. We also found that the α 2A AR was phosphorylated relatively well in the absence of epinephrine and transfected GRK2 suggesting that α 2A AR is a good substrate for an endogenous COS‐7 cell kinase. Mutagenesis of the receptor has allowed us to measure agonist‐ and GRK‐dependent phosphorylation of α 2A AR. We are now in a position to characterize GRK2 phosphorylation of this receptor.