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J. Biol. Chem., Vol. 283, Issue 35, 24000-24010, August 29, 2008

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The Ubiquitin-Protein Ligase Nedd4-2 Differentially Interacts with and Regulates Members of the Tweety Family of Chloride Ion Channels^*

Yaowu He, Deanne H. Hryciw¹, Melanie L. Carroll², Stephen A. Myers³, Astrid K. Whitbread, Sharad Kumar^¶, Philip Poronnik, and John D. Hooper⁴

From the Institute of Health and Biomedical Innovation and School of Life Sciences, Queensland University of Technology, Kelvin Grove, Queensland 4059, Australia, the School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia, and the ^¶Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, South Australia 5000, Australia

The Tweety proteins comprise a family of chloride ion channels with three members identified in humans (TTYH1-3) and orthologues in fly and murine species. In humans, increased TTYH2 expression is associated with cancer progression, whereas fly Tweety is associated with developmental processes. Structurally, Tweety proteins are characterized by five membrane-spanning domains and N-glycan modifications important for trafficking to the plasma membrane, where these proteins are oriented with the amino terminus located extracellularly and the carboxyl terminus cytoplasmically. In addition to N-glycosylation, ubiquitination mediated by the HECT type E3 ubiquitin ligase Nedd4-2 is a post-translation modification important in regulating membrane proteins. In the present study, we performed a comprehensive analysis of the ability of each of TTYH1-3 to interact with Nedd4-2 and to be ubiquitinated and regulated by this ligase. Our data indicate that Nedd4-2 binds to two family members, TTYH2 and TTYH3, which contain consensus PY ((L/P)PXY) binding sites for HECT type E3 ubiquitin ligases, but not to TTYH1, which lacks this motif. Consistently, Nedd4-2 ubiquitinates both TTYH2 and TTYH3. Importantly, we have shown that endogenous TTYH2 and Nedd4-2 are binding partners and demonstrated that the TTYH2 PY motif is essential for these interactions. We have also shown that Nedd4-2-mediated ubiquitination of TTYH2 is a critical regulator of cell surface and total cellular levels of this protein. These data, indicating that Nedd4-2 differentially interacts with and regulates TTYH1-3, will be important for understanding mechanisms controlling Tweety proteins in physiology and disease.

Received for publication, May 2, 2008 , and in revised form, June 18, 2008.

^* This work was supported by National Health and Medical Research Council of Australia grants (to J. D. H., P. P., and S. K.) and a fellowship (to J. D. H.) and Australian Research Council grants (to P. P. and S. K.). 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 Present address: School of Biomedical and Health Sciences, Victoria University, St. Albans, Victoria 3021, Australia.

² Present address: School of Medicine, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Queensland 4102, Australia.

³ Present address: Institute for Molecular Bioscience, University of Queens-land, Brisbane, Queensland 4072, Australia.

⁴ To whom correspondence should be addressed: Queensland University of Technology, Corner Musk Ave. and Blamey St., Kelvin Grove, Queensland 4059, Australia. Tel.: 61-7-31386198; Fax: 61-7-3138-6030; E-mail: jd.hooper{at}qut.edu.au.

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