Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates

Abstract

The functional unit of the Na,K-ATPase consists of a catalytic ␣ subunit noncovalently linked with a glycoprotein subunit, ␤. Using ouabain binding assays and immunoprecipitation of rodent ␣͞␤ complexes, we show here that all six possible isozymes between three ␣ and two ␤ isoforms can be formed in Xenopus oocytes. Two isoformspecific differences in ␣͞␤ interactions are observed: (i) ␣1͞␤1 and ␣2͞␤2 complexes, in contrast to ␣1͞␤2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) ␤2 subunits must carry N-glycans to combine with ␣1 but not with ␣2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated ␤1 subunit comprising the ectodomain results in assembly with ␣1 and ␣2, but not with ␣3; the ␤2 ectodomain combines with ␣2 only. A chimera consisting of 81% and 19% of the ␣1 N terminus and ␣2 C terminus, respectively, behaves like ␣2 and coprecipitates with the ␤2 ectodomain. In contrast, the reciprocal chimera does not coprecipitate with the ␤2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase ␣ and ␤ subunits.The Na,K-ATPase is a heterodimer consisting of a 110-kDa ␣ subunit that is noncovalently linked with a glycoprotein ␤ subunit (protein core, 33-35 kDa). The ␣ subunit is the catalytic active part and bears the site for ATP hydrolysis, the binding sites for Na ϩ , K ϩ , and cardiac glycosides (1). The ␣ subunit spans the membrane several times. The ␤ subunit exposes a short N-terminal tail to the cytoplasm and the majority of its mass at the extracellular surface. It confers conformational stability to the ␣ subunit during or after assembly in the endoplasmic reticulum and is needed for routing of the enzyme to the plasma membrane (2-5).Three ␣ isoforms (␣1, ␣2, and ␣3) and two ␤ isoforms (␤1 and ␤2) have been identified in mammals and birds, each encoded by a separate gene (6). The ␣ isoforms show more than 80% homology even for very diverse species. The two ␤ isoforms have only about 40% amino acid sequence identity within the same species. Sequences encoding a mammalian ␤3 isoform have been deposited in the GenBank database. Both ␣ and ␤ isoforms display complex patterns of tissue-specific and developmental expression (7,8).All six possible ␣͞␤ isozymes are formed from the three ␣ and two ␤ isoforms of birds (9) and mammals (this work) in vitro, supporting the assumption that different isozymes exist in vivo (10, 11). Hybrid Na,K-pumps are readily formed from ␣1 and ␤1 subunits of virtually all species investigated (for a review, see ref. 12). The ␤1 isoform can even be replaced by the ␤ subunit of the H,K-pump to yield a functional Na,Kpump (13).Protein domains involved in ␣1͞␤1 interactions have only partially been identified. Deletion of the cytoplasmic domain and part of the transmembrane domain of chicken ␤1 does not disturb assembly as long as the truncated mutants are inserted into the membrane (14). Chimeric ␤1 subunits containing the N terminus and the membrane spanning domain...

Subunit Isoform Selectivity in Assembly of Na,K-ATPase α-β Heterodimers

“…7). These results are in agreement with previously reported disruption of ␣ 1 -␤ 2 , but not of the ␣ 1 -␤ 1 or ␣ 2 -␤ 2 complexes formed in Xenopus oocytes by Triton X-100 (44,45).…”

The Na+/K+ ATPase is required for septate junction function and epithelial tube-size control in theDrosophilatracheal system

“…However, given that all combinations of Na + /K + ATPase α and β subunits form functional ion pumps (Lemas et al, 1994;Schmalzing et al, 1991;Schmalzing et al, 1997), these results also raise the possibility that neither nrv2 isoform has specific functions and that any β subunit could substitute for Nrv2. We therefore tested whether either of the two other Drosophila Na + /K + ATPase β subunits, nrv1 (Sun and Salvaterra, 1995b) or nrv3 (CG8663), had tube-size or barrier activities.…”

Cell Adhesion, Polarity, and Epithelia in the Dawn of Metazoans

“…It is composed of ϳ370 amino acids, with the first 30 exposed to the cytosol, and 300-fold to form the extracellular portion that has 3 disulfide bonds. There are 3 N-glycosylation consensus sequences (NXS or NXT) in the extracellular domain (249,400). The polypeptide chain of the ␤-subunit weighs 32-35 kDa, and when fully glycosylated can reach an overall apparent molecular mass of 55-60 kDa.…”