Interaction with the Na,K-ATPase and Tissue Distribution of FXYD5 (Related to Ion Channel)

Irina Lubarski,Kaarina Pihakaski-Maunsbach,Steven J.D Karlish,Arvid B Maunsbach,Haim Garty

doi:10.1074/jbc.m506397200

Irina Lubarski, Kaarina Pihakaski-Maunsbach + Show 3 more

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https://doi.org/10.1074/jbc.m506397200

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Abstract

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,K-ATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365-370). The goal of this study was to determine whether FXYD5 can modulate the Na,K-ATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henle's loop. FXYD5 was effectively immunoprecipitated by antibodies to the alpha subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates alpha. Co-expressing FXYD5 with the alpha1 and beta1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabain-blockable outward current or as ouabain-sensitive (86)Rb(+) uptake. Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.

Highlights

Kinetics in vivo and function as tissue-specific modulators of the Na,KATPase (9 –11)
In order to verify that the antibodies react with FXYD5, we have tested them on Xenopus oocytes injected with FXYD5 and Hemagglutinin A (HA)-FXYD5 cRNAs
In oocytes injected with HA-FXYD5, a similar 24-kDa polypeptide was detected by the anti-HA and anti-C antibodies

Summary

Introduction

Kinetics in vivo and function as tissue-specific modulators of the Na,KATPase (9 –11). FXYD proteins are type I membrane proteins with an extracellular N terminus (sometimes including a signal peptide), a single transmembrane domain, and an intracellular C terminus. With the exception of FXYD5, the extracellular domain is shorter than 40 amino acids, including a cleavable signal peptide. The apparent molecular mass of the protein detected in these studies is 50 –55 kDa, much higher than its calculated molecular mass (Ͻ20 kDa) This observation has been suggested to reflect an extensive O-glycosylation [14, 21]. This study reports the functional expression, some biochemical characterization, and the tissue and cellular distribution of FXYD5. Co-immunoprecipitation assays show that FXYD5 interacts with the ␣ subunit of Na, K-ATPase, and functional assays demonstrate effects on the pump kinetics. Part of these data have been reported in abstract form [22]

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