Structure-Function Relations of Interactions between Na,K-ATPase, the γ Subunit, and Corticosteroid Hormone-induced Factor

Several members of the FXYD protein family are tissue-specific regulators of Na,K-ATPase that produce distinct effects on its apparent K ؉ and Na ؉ affinity. Little is known about the interaction sites between the Na,KATPase ␣ subunit and FXYD proteins that mediate the efficient association and/or the functional effects of FXYD proteins. In this study, we have analyzed the role of the transmembrane segment TM9 of the Na,K-ATPase ␣ subunit in the structural and functional interaction with FXYD2, FXYD4, and FXYD7. Mutational analysis combined with expression in Xenopus oocytes reveals that Phe 956 , Glu 960 , Leu 964 , and Phe 967 in TM9 of the Na,K-ATPase ␣ subunit represent one face interacting with the three FXYD proteins. Leu 964 and Phe 967 contribute to the efficient association of FXYD proteins with the Na,K-ATPase ␣ subunit, whereas Phe 956 and Glu 960 are essential for the transmission of the functional effect of FXYD proteins on the apparent K ؉ affinity of Na,K-ATPase. The relative contribution of Phe 956 and Glu 960 to the K ؉ effect differs for different FXYD proteins, probably reflecting the intrinsic differences of FXYD proteins on the apparent K ؉ affinity of Na,K-ATPase. In contrast to the effect on the apparent K ؉ affinity, Phe 956 and Glu 960 are not involved in the effect of FXYD2 and FXYD4 on the apparent Na ؉ affinity of Na,K-ATPase. The mutational analysis is in good agreement with a docking model of the Na,K-ATPase/ FXYD7 complex, which also predicts the importance of Phe 956 , Glu 960 , Leu 964 , and Phe 967 in subunit interaction. In conclusion, by using mutational analysis and modeling, we show that TM9 of the Na,K-ATPase ␣ subunit exposes one face of the helix that interacts with FXYD proteins and contributes to the stable interaction with FXYD proteins, as well as mediating the effect of FXYD proteins on the apparent K ؉ affinity of Na,K-ATPase.

Section: Discussionsupporting

confidence: 78%

Structural and Functional Interaction Sites between Na,K-ATPase and FXYD Proteins

Grosdidier

Crambert

et al. 2004

“…As suggested by recent experimental evidence (33), the amino acids in FXYD proteins that are involved in the stable interaction with the Na,K-ATPase may not be the same as those involved in the functional effect, though they are also concentrated in the transmembrane domain. In our study, this could be reflected by the observation that some FXYD7 mutants affecting the N-terminal domain indeed decrease association efficiency in detergent but still produce the same functional effect on the K ϩ kinetics of Na,K-ATPase as wild type FXYD7.…”

Section: Discussionmentioning

confidence: 98%

“…The precise domain(s) or amino acids in FXYD proteins that are directly involved in the interaction with Na,K-ATPase are still unknown. Recent studies suggest that, both in FXYD2 and FXYD4, the transmembrane domain is mainly involved in the stable interaction with Na,K-ATPase (19,33) though in FXYD2 the cytoplasmic C terminus may also play a role in this process (8). In the present study, we confirm recent observations (19) that the conserved Gly 40 , which is associated with a form of renal hypomagnesemia when mutated into an arginine residue in FXYD2 (18), is involved in Na,K-ATPase-FXYD protein interaction.…”

Section: Discussionmentioning

confidence: 99%

FXYD7, Mapping of Functional Sites Involved in Endoplasmic Reticulum Export, Association With and Regulation of Na,K-ATPase

Crambert

Swee

et al. 2004

The brain-specific FXYD7 is a member of the recently defined FXYD family that associates with the ␣1-␤1 Na,K-ATPase isozyme and induces an about 2-fold decrease in its apparent K ؉ affinity. By using the Xenopus oocyte as an expression system, we have investigated the role of conserved and FXYD7-specific amino acids in the cellular routing of FXYD7 and in its association with and regulation of Na,K-ATPase. In contrast to FXYD2 and FXYD4, the studies on FXYD7 show that the conserved FXYD motif in the extracytoplasmic domain is not involved in the efficient association of FXYD7 with Na,K-ATPase. On the other hand, the conserved Gly 40 and Gly 29 , located on the same face of the transmembrane helix, were found to be implicated both in the association with and the regulation of Na,K-ATPase. Mutational analysis of FXYD7-specific regions revealed the presence of an ER export signal at the end of the cytoplasmic tail. Deletion of a C-terminal valine residue in FXYD7 significantly delayed and decreased its O-glycosylation processing and retarded the rate of its cell surface expression. This result indicates that the C-terminal valine residue is involved in the rapid and selective ER export of FXYD7, which could explain the observed post-translational association of FXYD7 with Na,KATPase. In conclusion, our study on FXYD7 provides new information on structural determinants of general importance for FXYD protein action. Moreover, FXYD7 is identified as a new member of proteins with a regulated ER export, which suggests that, among FXYD proteins, FXYD7 has a particular regulatory function in brain.

“…For immunocytochemistry, we have used antibodies that were purified by affinity chromatography using the immunizing peptides coupled to the HiTrap N-hydroxysuccinimideactivated HP column (Amersham Biosciences). Polyclonal antibodies to the C-terminal sequences of CHIF and ␥ were described previously (24). A monoclonal antibody to a sequence near the N terminus of the ␣1 subunit of Na,K-ATPase (6H) was kindly provided by Dr. M. J. Caplan, Yale University School of Medicine.…”

Section: Methodsmentioning

confidence: 99%

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

Lubarski

Pihakaski-Maunsbach

Karlish

et al. 2005

Self Cite

132

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,KATPase 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,KATPase 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 ␣ subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates ␣. Co-expressing FXYD5 with the ␣1 and ␤1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabainblockable 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.Work in several laboratories led to the identification of a family of proteins, named after the common motif FXYD (1). Five members of this group have been shown to interact with the Na,K-ATPase and elicit different effects on its kinetics. These are as follows: FXYD1 (phospholemman, PLM), 3 (2); FXYD2 (the ␥ subunit of Na,K-ATPase, ␥) (3); FXYD3 (Mat-8) (4); FXYD4 (corticosteroid hormone-induced factor, CHIF) (5); and FXYD7 (6). In addition, a PLM-like protein from shark rectal gland has been characterized (7,8). The remaining two family members FXYD5 (related to ion channel, dysadherin) and FXYD6 have not yet been analyzed for possible interactions with the Na,K-ATPase. The working hypothesis is that all family members modulate the pump kinetics in vivo and function as tissue-specific modulators of the Na,KATPase (9 -11). However, other functions for FXYD proteins have also been suggested (12-16).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. In the case of FXYD5, the extracellular domain is long, Ͼ140 amino acids. On the other hand, FXYD5 has the shortest intracellular C-terminal segment of only 15 amino acids. FXYD5 has been cloned as a tissue-specific and developmentally regulated gene induced by the oncoprotein E2a-Pbx1 and termed "related to ion channel...