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J. Biol. Chem., Vol. 283, Issue 21, 14516-14523, May 23, 2008

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Protein Kinase A Phosphorylation of Spinophilin Modulates Its Interaction with the _2A-Adrenergic Receptor (AR) and Alters Temporal Properties of _2AAR Internalization^*

Jianmin Xu, Yunjia Chen, Roujian Lu, Christopher Cottingham, Kai Jiao, and Qin Wang¹

From the Departments of Physiology and Biophysics and Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294

Spinophilin plays critical roles in regulating trafficking and signaling of the ₂-adrenergic receptor (AR) both in vitro and in vivo (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940–1944). In the present study, we demonstrate that protein kinase A (PKA) phosphorylation of spinophilin modulates the spinophilin-_2AAR interaction to regulate _2AAR internalization. Activation of PKA by forskolin abolishes the agonist-enhanced interaction between spinophilin and the _2AAR, and this event can be blocked by Ser Ala mutations at the PKA phosphorylation sites of spinophilin. In addition, a Ser Asp mutation that mimics the phosphorylated state at the PKA phosphorylation site Ser-177, which is located within the _2AAR binding region of spinophilin, is sufficient to block the spinophilin-_2AAR interaction in intact cells. In cells expressing mutant spinophilin carrying the S177D mutation, agonist-induced internalization of the _2AAR is accelerated and enhanced, as revealed by both intact cell enzyme-linked immunosorbent assay and quantitative immunofluorescent studies. Furthermore, activation of PKA by forskolin enhances agonist-induced internalization of the _2AAR in cells expressing wild type spinophilin, but not in cells lacking spinophilin or expressing the spinophilin mutant Sp177D. These results strongly support that PKA phosphorylation of spinophilin is functionally relevant in regulating _2AAR trafficking. Therefore, modulation of spinophilin-receptor interaction through phosphorylation of spinophilin may represent a novel mechanism whereby PKA regulates G protein-coupled receptor trafficking.

Received for publication, December 19, 2007 , and in revised form, March 20, 2008.

^* This work was supported by an Alabama state fund and an American Heart Association Scientist Development grant (to Q. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 958 MCLM, 1918 University Blvd., Birmingham, AL 35294. Tel.: 205-996-5099; Fax: 205-975-9028; E-mail: qinwang{at}uab.edu.

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