Abstract

The α2A‐adrenergic receptor (α2A‐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 α2A‐AR serves as an excellent substrate for agonist‐dependent phosphorylation by G protein‐coupled receptor (GPCR) kinase 2 (GRK2) in vitro. α2A‐AR is structurally distinct from the well‐characterized β2‐adrenergic receptor (β2AR) 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 β2AR and recruitment to α2A‐AR in intact cells. We wish to assay phosphorylation of α2A‐AR in intact cells and because no commercially available α2A‐AR phosphorylation site antibodies currently exist, we set out to develop such a reagent. An antibody that recognizes α2A‐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 α2A‐AR was phosphorylated relatively well in the absence of epinephrine and transfected GRK2 suggesting that α2A‐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 α2A‐AR. We are now in a position to characterize GRK2 phosphorylation of this receptor.Support or Funding InformationThis work was funded by the National Science Foundation MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).

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