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J. Biol. Chem., Vol. 282, Issue 39, 28721-28732, September 28, 2007

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Heat Shock Protein 70 Interacts with Aquaporin-2 and Regulates Its Trafficking^*

From the Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.

Received for publication, December 4, 2006 , and in revised form, July 6, 2007.

^* This work was supported in part by NIDDK Grant DK38452 from the National Institutes of Health (to D. B.). 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.

² Recipient of fellowship support from the National Kidney Foundation.

¹ Supported by National Institutes of Health K08 Award DK075940. To whom correspondence may be addressed: Program in Membrane Biology/Division of Nephrology, Massachusetts General Hospital, CPZN 1850, 185 Cambridge St., Boston, MA 02114. Tel.: 617-724-9694; Fax: 617-643-3182; E-mail: halu{at}partners.org.

³ To whom correspondence may be addressed. E-mail: brown{at}receptor.mgh.harvard.edu.

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