Correlation of Gene and Protein Structures in the FXYD Family Proteins

Abstract

The FXYD family proteins are auxiliary subunits of the Na,K-ATPase, expressed primarily in tissues that specialize in fluid or solute transport, or that are electrically excitable. These proteins range in size from about 60 to 160 amino acid residues, and share a core homology of 35 amino acid residues in and around a single transmembrane segment. Despite their relatively small sizes, they are all encoded by genes with six to nine small exons. We show that the helical secondary structures of three FXYD family members, FXYD1, FXYD3, and FXYD4, determined in micelles by NMR spectroscopy, reflect the structures of their corresponding genes. The coincidence of helical regions, and connecting segments, with the positions of intron-exon junctions in the genes, support the hypothesis that the FXYD proteins may have been assembled from discrete structural modules through exon shuffling. KeywordsFXYD; Na,K-ATPase; NMR structure; membrane protein; gene The FXYD family proteins are tissue-specific and physiological-state-specific auxiliary subunits of the Na, K-ATPase, the primary enzyme responsible for maintaining the equilibrium of Na and K ion concentrations across animal cell membranes. [1][2][3] All of the FXYD genes are expressed in the early stages of fetal life, with prevalence in tissues that specialize in fluid or solute transport, or that are electrically excitable. In keeping with their distribution in tissues, several FXYD family members have been shown to regulate ion transport by binding and modulating the activity of Na, K-ATPase molecules. [2][3][4][5][6][7][8][9][10][11] In addition, certain FXYD family members can induce ionic currents in Xenopus oocytes, or in phospholipid bilayers, although the direct formation of ion channels has not been demonstrated in vivo. [12][13][14][15][16] Seven FXYD family members have been identified in mammals. FXYD1 (PLM; phospholemman) is the principal substrate of hormone-stimulated phosphorylation by cAMP-dependent protein kinase A and C in sarcolemma heart. 17 FXYD2 (gamma), unique in the family for having two alternative splice variants (FXYD2a and FXYD2b), and FXYD4 (CHIF; channel-inducing factor; corticosteroid hormone induced factor), are each expressed in distinct, specialized segments of the kidney, with unique expression patterns that help explain the physiological differences in Na,K-ATPase activity among the nephron segments. 5,6,13,18,19 Two family members, FXYD3 (Mat8; mammary tumor protein 8 kDa) and FXYD5 (RIC; resembles ion channel; dyshaderin), are expressed in cancers and play a role in tumor progression. [20][21][22][23] and are believed to play a role in neuron excitability during postnatal development and in the adult brain. 8,24 Membrane proteins belonging to what is now recognized as the FXYD family were first discovered, in association with the Na,K-ATPase, 4 or as a major substrate of hormonestimulated phosphorylation in sarcolemma. 25 The subsequent sequencing and identification of additional proteins, with similar structural and functi...

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

“…The data suggest that the peaks in the PISEMA spectrum will have to be fitted with Pisa wheels of different tilts, in agreement with the solution NMR studies in micelles, showing that helices 1 and 2 have different orientations. According to the data in micelles, the peaks in the spectrum of 15 N-Leu labeled CHIF should account for Leu 17 and 19 in helix 1 preceding the transmembrane helix (helix 2), and Leu 22,27,28,35,37 in the transmembrane helix.…”

“…An important finding of these NMR structural studies was that the helical secondary structures of the FXYD family members, PLM, gamma, Mat-8, and CHIF, reflect the structures of their corresponding genes [28]. Despite their relatively small sizes (60-160 residues), the FXYD family proteins are all encoded by genes with six to nine small exons, and the coincidence of intron-exon junctions with helical structures and flexible connecting segments, support the hypothesis that the FXYD proteins were assembled from discrete structural modules through exon shuffling.…”

“…FXYD3 (Mat8; mammary tumor protein 8 kDa) is expressed in cancers and plays a role in tumor progression [26,27]. Despite their relatively small sizes ranging from about 60-160 amino acids, all FXYD proteins are encoded by genes with six to nine small exons, and NMR has shown that the protein structures reflect the structures of their corresponding genes, suggesting that they were assembled from modules through exon shuffling [28].…”

Nuclear Magnetic Resonance Structural Studies of Membrane Proteins in Micelles and Bilayers

“…An important finding of these structural studies is that the helical secondary structures of the FXYD family proteins reflect the structures of their corresponding genes (18). The coincidence of intron-exon junctions with helical structures and flexible connecting segments, support the hypothesis that the FXYD proteins may have been assembled from discrete structural modules through exon shuffling.…”

Down-Regulation of FXYD3 Expression in Human Lung Cancers

“…and not a rare polymorphism ( Figure 3A). Asparate at the 19th amino acid (D19), part of the first transmembrane helix of FXYD3, is evolutionally conserved (Figure 3B), 9,24,25 and likely to be essential to its function.…”