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Originally published In Press as doi:10.1074/jbc.M011679200 on January 11, 2001

J. Biol. Chem., Vol. 276, Issue 18, 15003-15008, May 4, 2001
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Agonist-regulated Interaction between alpha 2-Adrenergic Receptors and Spinophilin*

Jeremy G. RichmanDagger §, Ashley E. BradyDagger , Qin WangDagger , Jennifer L. HenselDagger , Roger J. Colbran||, and Lee E. LimbirdDagger **

From the Departments of Dagger  Pharmacology and of || Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600

Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical alpha 2A-adrenergic receptor (alpha 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 alpha 2AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the alpha 2AAR (Val217-Ala377), alpha 2BAR (Lys210-Trp354), and alpha 2CAR (Arg248-Val363) 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 alpha 2AAR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to alpha 2AR localization but also to agonist modulation of alpha 2AR signaling.

* This work was supported in part by National Institutes of Health Grants DK43879 (to L. E. L.) and NS37508 (to R. J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Funded by Postdoctoral Training Grant T32DK07563 during part of these studies.

Funded by an advanced pre-doctoral Fellowship in Pharmacology and Toxicology from the Pharmaceutical Research and Manufacturers Assn. Foundation. Confocal microscopy was performed in the Vanderbilt University Medical Center Cell Imaging Core Resource Facility (supported by NIH Grants CA68485 and DK20593).

** To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, Rm. 464, Nashville, TN 37232-6600. Tel.: 615-343-3538; Fax: 615-343-1084; E-mail: Lee. Limbird@mcmail.vanderbilt.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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