Spinophilin plays critical roles in regulating trafficking and signaling of the α2-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-α2AAR interaction to regulate α2AAR internalization. Activation of PKA by forskolin abolishes the agonist-enhanced interaction between spinophilin and the α2AAR, 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 α2AAR binding region of spinophilin, is sufficient to block the spinophilin-α2AAR interaction in intact cells. In cells expressing mutant spinophilin carrying the S177D mutation, agonist-induced internalization of the α2AARis 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 α2AAR 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 α2AAR trafficking. Therefore, modulation of spinophilin-receptor interaction through phosphorylation of spinophilin may represent a novel mechanism whereby PKA regulates G protein-coupled receptor trafficking. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.