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J. Biol. Chem., Vol. 279, Issue 29, 30410-30418, July 16, 2004

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Clathrin-mediated Endocytosis of m₃ Muscarinic Receptors

ROLES FOR G AND TUBULIN^*

Juliana S. Popova and Mark M. Rasenick¶

From the Departments of Physiology and Biophysics and ^¶Psychiatry, College of Medicine, University of Illinois, Chicago, Illinois 60612-7342

Receptors as well as some G protein subunits internalize after agonist stimulation. It is not clear whether G_q or G undergo such regulated translocation. Recent studies demonstrate that m₃ muscarinic receptor activation in SK-N-SH neuroblastoma cells causes recruitment of tubulin to the plasma membrane. This subsequently transactivates G_q and activates phospholipase C₁. Interaction of tubulin-GDP with G at the offset of phospholipase C₁ signaling appears involved in translocation of tubulin and G to vesicle-like structures in the cytosol (Popova, J. S., and Rasenick, M. M. (2003) J. Biol. Chem. 278, 34299–34308). The relationship of this internalization to the clathrin-mediated endocytosis of the activated m₃ muscarinic receptors or G_q involvement in this process has not been clarified. To test this, SK-N-SH cells were treated with carbachol, and localization of G_q, G, tubulin, clathrin, and m₃ receptors were analyzed by both cellular imaging and biochemical techniques. Upon agonist stimulation both tubulin and clathrin translocated to the plasma membrane and co-localized with receptors, G_q and G. Fifteen minutes later receptors, G and tubulin, but not G_q, internalized with the clathrin-coated vesicles. Coimmunoprecipitation of m₃ receptors with G, tubulin, and clathrin from the cytosol of carbachol-treated cells was readily observed. These data suggested that G subunits might organize the formation of a multiprotein complex linking m₃ receptors to tubulin since they interacted with both proteins. Such protein assemblies might explain the dynamin-dependent but -arrestin-independent endocytosis of m₃ muscarinic receptors since tubulin interaction with dynamin might guide or insert the complex into clathrin-coated pits. This novel mechanism of internalization might prove important for other -arrestin-independent endocytic pathways. It also suggests cross-regulation between G protein-mediated signaling and the dynamics of the microtubule cytoskeleton.

Received for publication, March 15, 2004 , and in revised form, April 22, 2004.

^* This study was supported by National Institutes of Health Grants MH 39595 and AG 15482 (to M. M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave. M/C 901, Chicago, IL 60612-7342. Tel.: 312-996-6641; Fax: 312-996-1414; E-mail: jsp{at}uic.edu.

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