Abstract
Six of 7 FXYD proteins have been shown to be tissue-specific modulators of Na,K-ATPase. In this study, we have identified two splice variants of human FXYD3, or Mat-8, in CaCo-2 cells. Short human FXYD3 has 72% sequence identity with mouse FXYD3, whereas long human FXYD3 is identical to short human FXYD3 but has a 26-amino acid insertion after the transmembrane domain. Short and long human FXYD3 RNAs and proteins are differentially expressed during differentiation of CaCo-2 cells. Long human FXYD3 is mainly expressed in nondifferentiated cells and short human FXYD3 in differentiated cells and both FXYD3 variants can be co-immunoprecipitated with a Na,K-ATPase antibody. In contrast to mouse FXYD3, which has two transmembrane domains for lack of cleavage of the signal peptide, human FXYD3 has a cleavable signal peptide and adopts a type I topology. After co-expression in Xenopus oocytes, both human FXYD3 variants associate stably only with Na,K-ATPase isozymes but not with H,K-ATPase or Ca-ATPase. Similar to mouse FXYD3, short human FXYD3 decreases the apparent K(+) and Na(+) affinity of Na,K-ATPase over a large range of membrane potentials. On the other hand, long human FXYD3 decreases the apparent K(+) affinity only at slightly negative and positive membrane potentials and increases the apparent Na(+) affinity of Na,K-ATPase. Finally, both short and long human FXYD3 induce a hyperpolarization activated current, similar to that induced by mouse FXYD3. Thus, we have characterized two human FXYD3 isoforms that are differentially expressed in differentiated and non-differentiated cells and show different functional properties.
Highlights
Segments and an ATP-hydrolyzing domain and is responsible for ion transport
The sequence coding for the additional 26 amino acids of FXYD3-lf was PCR amplified by using a sense primer tailed with 15 base pairs coding for the last amino acids of the N-terminal part of FXYD1/FXYD2 (5Ј-CTCATCGTCCTGAGCGAGTGGAGGAGCTCGGGGGAG-3Ј/5Ј-CTCATCCTCCTCAGCGAGTGGAGGAGCTCGGGGGAG-3Ј) and an antisense primer tailed with 15 base pairs coding for the first amino acids of the C-terminal part of FXYD1/FXYD2 (5Ј-TTTGCACCGGCATCTTCTACCTGTTGGGGAGAGCTG-3Ј/5Ј-CCCACAGCGGAATCTTCTACCTGTTGGGGAGAGCTG-3Ј)
The FXYD3-lf protein was the major form in non-differentiated cells and its expression slightly decreased during differentiation, whereas the FXYD3-sf protein was the major form in differentiated cells and its expression increased during differentiation (Fig. 1E)
Summary
Segments and an ATP-hydrolyzing domain and is responsible for ion transport. The  subunit that is a type II, glycosylated protein, plays a chaperone role by allowing the structural and functional maturation of Na,K-ATPase (1). It has been shown that FXYD3 mRNA expression is increased after treatment of MCF-7 breast cancer cells with the fluoropyrimidine drug, 5-fluorouracil, and that this activation is dependent on p53 expression (19) It remains to be shown whether there is a correlation between the regulation of the functional properties of Na,K-ATPase by FXYD3 and the possible role of FXYD3 in cell proliferation. We have identified two human FXYD3 isoforms in CaCo-2 cells that are differentially expressed during cell differentiation and are expressed in the MCF-7 breast cancer cell line Both human FXYD3 isoforms are able to stably associate with Na,K-ATPase and play different functional roles in the regulation of Na,K-ATPase activities
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
