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Intact Cell Assay to Measure Agonist‐ and GRK2‐Dependent Phosphorylation of the α 2A ‐Adrenergic Receptor
Author(s) -
Farina Zachary,
Hebert AaronMichael,
Weldin Erica,
Varney Matthew,
SterneMarr Rachel
Publication year - 2018
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.2018.32.1_supplement.533.32
Subject(s) - beta adrenergic receptor kinase , phosphorylation , g protein coupled receptor , agonist , g protein coupled receptor kinase , receptor , adrenergic receptor , serine , chemistry , microbiology and biotechnology , biology , biochemistry
The α 2A ‐adrenergic receptor (α 2A ‐AR) is a G protein‐coupled receptor (GPCR) that is activated by the catecholamines epinephrine and norepinephrine. Its physiological roles include inhibition of insulin secretion from pancreatic beta cells, cardiovascular vasoconstriction, and the inhibition of lipolysis in adipocytes (among other functions). GPCRs are often phosphorylated in an agonist‐dependent fashion and thereby desensitized by GPCR kinases (GRKs). GRK2 phosphorylates the α 2A ‐AR in vitro and when co‐transfected with the receptor in monkey kidney (COS‐7) cells. GRK2 phosphorylation occurs at four consecutive serine residues in the third intracellular loop of the α 2A ‐AR (residues 296–299). We recently mapped sites on GRK2 that are necessary for phosphorylation of the β 2 ‐adrenergic receptor and interaction with the α 2A ‐adrenergic receptor in intact cells. In order to test GRK2 mediated α 2A ‐AR phosphorylation, we generated an antibody that detects α 2A ‐AR phosphorylation at serines 296–299. We are developing an intact cell assay to assess residues on both GRK2 and the receptor that are important for α 2A ‐AR phosphorylation. This will give us a greater understanding of the regulation of this physiologically important receptor. Support or Funding Information This work was funded by the National Science Foundation MCB0744739 to Rachel Sterne‐Marr and the Siena College Center for Undergraduate Research and Creative Activity. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .