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J. Biol. Chem., Vol. 283, Issue 12, 7674-7681, March 21, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/12/7674    most recent M705574200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Lin, D. Articles by Wang, W.-H. PubMed PubMed Citation Articles by Lin, D. Articles by Wang, W.-H. Social Bookmarking                      What's this?

Expression of Tetraspan Protein CD63 Activates Protein-tyrosine Kinase (PTK) and Enhances the PTK-induced Inhibition of ROMK Channels^*

Daohong Lin, Erik-Jan Kamsteeg, Yan Zhang, Yan Jin, Hyacinth Sterling, Peng Yue, Marcel Roos, Amy Duffield, Joanna Spencer, Michael Caplan, and Wen-Hui Wang¹

From the Department of Pharmacology, New York Medical College, Valhalla, New York 10595 and the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase (RPTP) are expressed in the cortical collecting duct and outer medulla collecting duct. Immunoprecipitation of tissue lysates from renal cortex and outer medulla or 293T cells transfected with CD63 reveals that CD63 was associated with RPTP both in situ and in transfected cells. Expression of CD63 in 293T cells stimulated the phosphorylation of tyrosine residue 416 of c-Src but decreased the phosphorylation of tyrosine residue 527, indicating that expression of CD63 stimulates the activity of c-Src. Furthermore, c-Src was coimmunoprecipitated with RPTP and CD63 both in 293T cells transfected with CD63 and in lysates prepared from native rat kidney. Potassium restriction had no effect on the expression of RPTP, but it increased the association between c-Src and RPTP in the renal cortex and outer medulla. We also used two-electrode voltage clamp to study the effect of CD63 on ROMK channels in Xenopus oocytes. Expression of CD63 had no significant effect on potassium currents in oocytes injected with ROMK1; however, it significantly enhanced the c-Src-induced inhibition of ROMK channels in oocytes injected with ROMK1+c-Src. The effect of CD63 on the c-Src-induced inhibition was not due to a decreased expression of ROMK1 channels, because blocking PTK with herbimycin A abolished the inhibitory effect of c-Src on ROMK channels in oocytes injected with ROMK1+c-Src+CD63. Furthermore, coexpression of CD63 enhanced tyrosine phosphorylation of ROMK1. We conclude that CD63 plays a role in the regulation of ROMK channels through its association with RPTP, which in turn interacts with and activates Src family PTK, thus reducing ROMK activity.

Received for publication, July 6, 2007 , and in revised form, January 10, 2008.

^* This work was supported by National Institutes of Health Grants DK54983 (to W.-H. W.) and DK17433 (to M. C.). 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. Tel.: 914-594-4139; Fax: 914-347-4956; E-mail: wenhui_wang{at}nymc.edu.

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