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J. Biol. Chem., Vol. 282, Issue 32, 23296-23305, August 10, 2007

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Role of the Guanine Nucleotide Exchange Factor Ost in Negative Regulation of Receptor Endocytosis by the Small GTPase Rac1^*

From the Division of Molecular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan

The Rho family of GTPases has been implicated in the regulation of intracellular vesicle trafficking. Here, we investigated the mechanism underlying the negative regulation of clathrin-mediated endocytosis of cell surface receptors mediated by the Rho family protein Rac1. Contrary to previous reports, only the activated mutant of Rac1, but not other Rho family members including RhoA and Cdc42, suppressed internalization of the transferrin receptor. On the other hand, down-regulation of Rac1 expression by RNA interference resulted in enhanced receptor internalization, suggesting that endogenous Rac1 in fact functions as a negative regulator. We identified a guanine nucleotide exchange factor splice variant designated Ost-III, which contains a unique C-terminal region including an Src homology 3 domain, as a regulator of Rac1 involved in the inhibition of receptor endocytosis. In contrast, other splice variants Ost-I and Ost-II exerted virtually no effect on receptor endocytosis. We also examined subcellular localization of synaptojanin 2, a putative Rac1 effector implicated in negative regulation of receptor endocytosis. Each Ost splice variant induced distinct subcellular localization of synaptojanin 2, depending on Rac1 activation. Furthermore, we isolated -aminobutyric acid type A receptor-associated protein (GABARAP) as a protein that binds to the C-terminal region of Ost-III. When ectopically expressed, GABARAP was co-localized with Ost-III and potently suppressed the Ost-III-dependent Rac1 activation and the inhibition of receptor endocytosis. Lipid modification of GABARAP was necessary for the suppression of Ost-III. These results are discussed in terms of subcellular region-specific regulation of the Rac1-dependent signaling pathway that negatively regulates clathrin-mediated endocytosis.

Received for publication, February 1, 2007 , and in revised form, June 5, 2007.

^* This research was supported by grants-in-aid for Scientific Research on Priority Areas "Systems Genomics" and "Life Surveyor," Scientific Research (B), and the 21st Century COE Research Program "Center of Excellence for Signal Transduction Disease: Diabetes Mellitus as Model" from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by grants from the Takeda Science Foundation and the Yasuda Medical Foundation. This research was also supported by a grant for Initiatives for Attractive Education in Graduate Schools "Program of Raising Young Research Leaders in Biomedical Sciences" from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 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.

¹ To whom correspondence should be addressed: Division of Molecular Biology, Dept. of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel.: 81-78-382-5381; Fax: 81-78-382-5399; E-mail: tkysato{at}med.kobe-u.ac.jp.

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