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α 2A ‐Adrenergic Receptor: Expression in Insect Cells and Detection of Phosphorylation
Author(s) -
Varney Matt,
Hebert Aaron,
Vijay Jesus,
Kaszubowski Joe,
Beer Jacob,
Donahue Ryan,
SterneMarr Rachel
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.710.14
Subject(s) - phosphorylation , receptor , g protein coupled receptor , phosphatase , serine , beta adrenergic receptor kinase , microbiology and biotechnology , chemistry , adrenergic receptor , alanine , signal transduction , kinase , biochemistry , biology , amino acid
The α 2A ‐adrenergic receptor (α 2A AR) is a member of the G‐protein coupled receptor (GPCR) superfamily that responds to catecholamines such as epinephrine. In contrast to β‐adrenergic receptors, α 2 ‐adrenergic receptors act to decrease blood pressure and control pain perception during the “flight or fight” response. GPCR kinases (GRKs) are responsible for deactivating these receptors by phosphorylating their third intracellular loop or carboxyl terminal tail peptide. The phosphate group attracts proteins known as arrestins that terminate the signal. GRK2 has been shown to have a critical role in desensitization of the β 2 ‐adrenergic receptor and other receptors but its role in regulating the α 2A AR is less understood. Phosphorylation occurs on four consecutive serines (296‐299) in the third intracellular loop of the wild type (WT) α 2A AR. Previously in the lab, we generated a phosphopeptide antibody and therefore our goal was to determine the specificity of its recognition. First we showed that we could express WT α 2A AR using a baculovirus infection system. We then prepared a mutant baculovirus expressing α 2A AR with serines 296‐299 converted to alanine. The phosphorylation site antibody recognized the WT but not the mutant receptor. Furthermore, phosphatase treatment of phosphorylated WT α 2A AR removed the signal. This signal was maintained when the same experiment was carried out in the presence of phosphatase inhibitors thus demonstrating the specificity of the antibody for phosphorylated receptor. With a bona fide α 2A AR phosphorylation site antibody, GRK2 and its interaction with and phosphorylation of the α 2A AR can now be investigated.