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GPCR Kinase 2 (GRK2) Phosphosphorylation of the Alpha 2A Adrenergic Receptor in Intact Cells
Author(s) -
Sterne-Marr Rachel,
Barnett Jessica,
Hebert Aaron,
Weldin Erica,
Farina Zachary,
Guirguis Jeska,
Varney Matthew
Publication year - 2020
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.2020.34.s1.02922
Subject(s) - beta adrenergic receptor kinase , phosphorylation , g protein coupled receptor , chemistry , g protein coupled receptor kinase , microbiology and biotechnology , receptor , biochemistry , kinase , biology
The α 2A ‐adrenergic receptor (α 2A ‐AR) is activated by the catecholamine norepinephrine and plays a variety of 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 β 2 ‐adrenergic receptor (β 2 AR) in possessing a large (~100 amino acid) third intracellular loop containing four successive serine residues that serve as phospho‐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 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 recognizes 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. Support or Funding Information This work was funded by the National Science Foundation MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).