title: A basolateral sorting signal is encoded in the alpha-subunit of Na-K-ATPase.
abstract: Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that exhibit distinct plasma membrane distributions in epithelial cells. We have studied the alpha-subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric alpha-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the remaining 500 amino acids are derived from the H-K-ATPase sequence. In stably transfected LLC-PK1 cell lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within the NH2-terminal 519 amino acids of the Na-K-ATPase alpha-subunit contain a basolateral sorting signal. H-K-ATPase beta-subunit expressed alone in LLC-PK1 cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase beta-subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase alpha-subunit masks or is dominant over any apical sorting information present in the beta-polypeptide. In gastric parietal cells, the H-K-ATPase beta-subunit targets the H-K-ATPase to an intracellular vesicular compartment which fuses with the plasma membrane in response to secretagogue stimulation. To test whether the chimera-H-K-ATPase beta-subunit complex is directed to a similar compartment in LLC-PK1 cells, we treated transfected cells with drugs that raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Elevation of cytosolic cAMP increased the surface expression of both the N519H chimera and the H-K-ATPase beta-subunit. This increase in surface expression, however, appears to be the result of transcriptional upregulation and not recruitment of chimera to the surface from a cAMP-inducible compartment.
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: T R
authors.initials: TR
authors.lastname: Muth
authors.forename: C J
authors.initials: CJ
authors.lastname: Gottardi
authors.forename: D L
authors.initials: DL
authors.lastname: Roush
authors.forename: M J
authors.initials: MJ
authors.lastname: Caplan
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D016764:Cell Polarity; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D018374:LLC-PK1 Cells; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013499:Surface Properties; D013552:Swine; D014162:Transfection
chemical_list: D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D000242:Cyclic AMP; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D015056:1-Methyl-3-isobutylxanthine