Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark

Self Cite

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, Cys 254 . Thus, regulation of Na,KATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark ␣-subunit.

Section: Resultssupporting

confidence: 74%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Mahmmoud

Vorum

Cornelius³

2005

Self Cite

“…FXYD2 or the ␥ subunit of Na,K-ATPase (8) was the first FXYD protein that was identified as a specific modulator of renal Na,K-ATPase (9 -13). It is now well established that also FXYD1 (phospholemman) (14), a phospholemman-like protein from shark (15), FXYD4 (corticosteroid hormone-induced factor) (11,16), and FXYD7 (17) also play a tissue-specific role in Na,K-ATPase regulation. Significantly, each of these auxiliary subunits produce a distinct functional effect on Na,K-ATPase that is adapted to the physiological needs of the tissues in which they are expressed.…”

mentioning

confidence: 99%

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

Grosdidier

Crambert

et al. 2004

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.

“…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.…”

mentioning

confidence: 99%

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

Lubarski

Pihakaski-Maunsbach

Karlish

et al. 2005

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...