Protein Kinase A Phosphorylation of Spinophilin Modulates Its Interaction with the α2A-Adrenergic Receptor (AR) and Alters Temporal Properties of α2AAR Internalization

Abstract

The ␣ 2 adrenergic receptor (AR) 2 belongs to the G proteincoupled receptor superfamily and couples to the G i / o subfamily of G proteins. In native cells, activation of the ␣ 2 AR leads to inhibition of adenylyl cyclase and voltage-gated Ca 2ϩ channels and activation of inward rectifying K ϩ channels and mitogenactivated protein kinases (1, 2). Ligand stimulation also causes internalization of the ␣ 2 AR, a process that is important in regulating the sensitivity and duration of receptor-mediated signaling (3-5). ␣ 2 AR internalization is mediated by ␤-arrestins (6), which bind to the ␣ 2 AR (7-9) after the receptor is phosphorylated by G protein-coupled receptor kinases (10,11). In previous studies, we identified spinophilin as a competitor of G protein-coupled receptor kinase and ␤-arrestin 2 for binding to the third intracellular loop (3iloop) of the ␣ 2 AR (9, 12) and showed that interaction of spinophilin with the ␣ 2 AR is enhanced by agonist stimulation of the receptor (13). As a result, spinophilin stabilizes the ␣ 2 AR at the cell surface, as evident in cells without spinophilin expression, where internalization of the ␣ 2 AR is significantly accelerated and enhanced (12,14). In addition to all three ␣ 2 AR subtypes (␣ 2A , ␣ 2B , and ␣ 2C AR) (13,14), spinophilin interacts with several other G protein-coupled receptors, including the D2 dopamine receptor (15) and the ␣ 1 AR (16).Spinophilin is a multidomain protein containing an actin binding domain (amino acids (aa) 1-151), a protein phosphatase 1 binding sequence (aa 427-470), a PDZ domain (aa 496-586), and three coiled-coil domains at the C terminus (aa 607-817) (17, 18). The ␣ 2 AR interaction region of spinophilin is mapped to aa 151-444, which is adjacent to the actin binding domain (13). Spinophilin is a substrate of several protein kinases, including PKA (19), CaMKII (20), and ERK (21), suggesting that activities of spinophilin can be regulated by multiple intracellular pathways. PKA phosphorylation of spinophilin can be detected both in cultured cells and in the brain, and the target sites were mapped at . In the present study, we addressed whether phosphorylation of spinophilin by PKA affects its interaction with the ␣ 2A AR and investigated the functional relevance of phosphorylation of spinophilin at Ser-177 on the temporal properties of ␣ 2A AR internalization. Our studies suggest that PKA regulates ␣ 2A AR trafficking through phosphorylation of spinophilin and subsequent blockade of the spinophilin-␣ 2A AR interaction, which represents a potential novel mechanism by which other intracellular pathways regulate ␣ 2A AR function. EXPERIMENTAL PROCEDURES MaterialsDulbecco's modified Eagle's medium (Invitrogen), fetal bovine serum, Lipofectamine 2000, and calcium phosphate transfection kit were purchased from Invitrogen. Restriction

Governing effect of regulatory proteins for Cl−/HCO3− exchanger 2 activity

“…Anion exchangers are fundamental to the function of various secretory organ systems such as fluid and SPL is a widely distributed multi-domain scaffold protein that binds several GPCRs, including a1-adrenoceptors, all a2-adrenoceptors, and TRPC5/6 channels, [21][22][23][24] suggesting that SPL is involved in multiple intracellular pathways. The discovery of partner proteins of SPL and its modulatory functions has persisted mostly in the field of neurobiology because SPL was characterized in rat brains.…”

PDZ Protein Regulation of G Protein–Coupled Receptor Trafficking and Signaling Pathways

“…Spinophilin functions to promote membrane localization and inhibit the endocytosis and desensitization of a 2 ARs by competing for b-arrestin2 binding (Wang et al, 2004b). The interaction between spinophilin and a 2 AR is prevented by PKA-mediated phosphorylation of spinophilin, which results in increased agonist-stimulated a 2A AR endocytosis (Xu et al, 2008). b 2 AR activation also stimulates PKA-mediated spinophilin phosphorylation to increase a 2A AR endocytosis (Cottingham et al, 2013).…”

The Antidepressant Desipramine Is an Arrestin-biased Ligand at the α2A-Adrenergic Receptor Driving Receptor Down-regulation in Vitro and in Vivo

“…Intact Cell Surface ELISA-Receptor internalization was quantitatively assessed using an intact cell surface ELISA method (44,45). Briefly, MEF cells (seeded onto 96-well culture plates at 1 ϫ 10 4 cells/well) were stimulated acutely with DMI or NE, and then subjected to fixation, blocking, primary antibody (HA11, 1:3000), and secondary antibody (HRP-conjugated anti-mouse, 1:2000).…”

“…Immunofluorescent Staining-Receptor internalization was qualitatively assessed with a pre-labeling method for staining of HA-tagged ␣ 2A ARs as previously described (33,45). MEF cells (seeded onto glass cover slips in 24-well culture plates at 2 ϫ 10 4 cells/well) were acutely stimulated with DMI or NE.…”