Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical α -adrenergic receptor (α AR) 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 D 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 α AR subtypes might also interact with spinophilin. [ S]Met-labeled 3i loops of the α AR (Val -Ala ), α AR (Lys -Trp ), and α AR (Arg -Val ) 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 AR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to α AR localization but also to agonist modulation of α AR signaling. 2A 2A 2 2 2A 2B 2C 2A 2 2 35 217 377 210 354 248 303