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α2A‐Adrenergic Receptor: Expression in Insect Cells and Detection of Phosphorylation

The α2A‐adrenergic receptor (α2AAR) is a member of the G‐protein coupled receptor (GPCR) superfamily that responds to catecholamines such as epinephrine. In contrast to β‐adrenergic receptors, α2‐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 β2‐adrenergic receptor and other receptors but its role in regulating the α2AAR is less understood. Phosphorylation occurs on four consecutive serines (296‐299) in the third intracellular loop of the wild type (WT) α2AAR. 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 α2AAR using a baculovirus infection system. We then prepared a mutant baculovirus expressing α2AAR 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 α2AAR 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 α2AAR phosphorylation site antibody, GRK2 and its interaction with and phosphorylation of the α2AAR can now be investigated.