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J. Biol. Chem., Vol. 280, Issue 30, 27776-27782, July 29, 2005

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Interaction of FXYD10 (PLMS) with Na,K-ATPase from Shark Rectal Glands

CLOSE PROXIMITY OF Cys⁷⁴ OF FXYD10 TO Cys²⁵⁴ IN THE A DOMAIN OF THE -SUBUNIT REVEALED BY INTERMOLECULAR THIOL CROSS-LINKING^*

Yasser Ahmed Mahmmoud, Henrik Vorum¶, and Flemming Cornelius

From the Institutes of Physiology and Biophysics and ^¶Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the -subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the -subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog ,-methyleneadenosine 5'-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the -subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the -subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the -subunit comprising 33 amino acids, including a single Cys residue, Cys²⁵⁴. Thus, regulation of Na,K-ATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark -subunit.

Received for publication, March 22, 2005 , and in revised form, April 28, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ781093.

^* This work was supported by postdoctoral fellowships from the Danish Research Academy (Grant 22-01-0499) and the Faculty of Health Sciences, University of Aarhus. 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: Institute of Physiology and Biophysics, Ole Worms Allé 185, DK-8000 Aarhus C, Denmark. Tel.: 45-89422927; Fax: 45-861-29599; E-mail: yam{at}biophys.au.dk.

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