Interaction of alpha- and beta-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase beta-subunit.
The assembly of the beta-subunit of the gastric H-K-ATPase (HKbeta) with the alpha-subunit of the H-K-ATPase or the Na-K-ATPase (NaKalpha) was characterized with two anti-HKbeta monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells transfected with HKbeta, MAb 2/2E6 was observed to bind to HKbeta only when interactions between alpha- and beta-subunits were disrupted by various denaturants. The epitope for MAb 2/2E6 was mapped to the tetrapeptide S(226)LHY(229) of the extracellular domain of HKbeta. The epitope for MAb 2G11 was mapped to the eight NH(2)-terminal amino acids of the cytoplasmic domain of HKbeta. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HKbeta with alpha-subunits of the endogenous cell surface NaKalpha, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HKbeta. In HKbeta-transfected LLC-PK(1) cells, significant immunofluorescent labeling of HKbeta at the cell surface could be detected without postfixation denaturation or in live cells, although a fraction of transfected HKbeta could also be coimmunoprecipitated with NaKalpha. Thus assembly of HKbeta with NaKalpha does not appear to be a stringent requirement for cell surface delivery of HKbeta in LLC-PK(1) cells but may be required in MDCK cells. In addition, endogenous posttranslational regulatory mechanisms to prevent hybrid alpha-beta heterodimer assembly appear to be compromised in transfected cultured renal epithelial cells. Finally, the extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a region of HKbeta that is associated with alpha-beta interaction.
Okamoto
C T
CT
Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90089-9121, USA. cokamoto@hsc.usc.edu
Chow
D C
DC
Forte
A J
AJ
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004285:Dogs; D000939:Epitopes; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008565:Membrane Proteins / Q000378:metabolism; D010295:Parietal Cells, Gastric / Q000201:enzymology; D010446:Peptide Fragments / Q000276:immunology; D010449:Peptide Mapping; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007527:Isoenzymes; D008565:Membrane Proteins; D010446:Peptide Fragments; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase