Agonist-regulated interaction between alpha(2)-adrenergic receptors and spinophilin

Previously, we demonstrated that the third intracellular (3i) loop of the h eptahelical alpha (2A)-adrenergic receptor (alpha (2A)AAR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface, Findin gs that the 3i loops of the D-2 dopamine receptors interact with spinophili n (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999) J. Biol. Chem. 27 4, 19894-19900) and that spinophilin is enriched beneath the basolateral su rface of polarized MDCK cells prompted us to assess whether alpha (2)AR sub types might also interact with spinophilin. [S-35]Met-labeled 3i loops of t he alpha (2A)AR (Val(217)-Ala(377)), alpha (2B)AR (Lys(210)-Trp(354)), and alpha (2C)AR (Arg(248)-Val(363)) subtypes interacted with glutathione S-tra nsferase-spinophilin fusion proteins. These interactions could be refined t o spinophilin amino acid residues 169-255, in a region between spinophilin' s F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist regulated fashion, because alpha (2A)AR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin ma y contribute not only to alpha (2)AR localization but also to agonist modul ation of alpha (2)AR signaling.