Agonist-regulated Interaction between α₂-Adrenergic Receptors and Spinophilin

Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical α_2A-adrenergic receptor (α_2AAR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface. Findings that the 3i loops of the D₂ dopamine receptors interact with spinophilin (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999) J. Biol. Chem. 274, 19894-19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether α₂AR subtypes might also interact with spinophilin. [³⁵S]Met-labeled 3i loops of the α_2AAR (Val ²¹⁷-Ala³⁷⁷), α_2BAR (Lys ²¹⁰-Trp³⁵⁴), and α_2CAR (Arg ²⁴⁸-Val³⁰³) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to 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 α_2AAR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to α₂AR localization but also to agonist modulation of α₂AR signaling.