title: Calcineurin B homologous protein 3 promotes the biosynthetic maturation, cell surface stability, and optimal transport of the Na+/H+ exchanger NHE1 isoform.
abstract: Calcineurin B homologous protein (CHP) 1 and 2 are Ca(2+)-binding proteins that modulate several cellular processes, including cytoplasmic pH by positively regulating plasma membrane-type Na(+)/H(+) exchangers (NHEs). Recently another CHP-related protein, termed tescalcin or CHP3, was also shown to interact with the ubiquitous NHE1 isoform, but seemingly suppressed its activity. However, the precise physical and functional nature of this association was not examined in detail. In this study, biochemical and cellular studies were undertaken to further delineate this relationship. Glutathione S-transferase-NHE1 fusion protein pulldown assays revealed that full-length CHP3 binds directly to the proximal juxtamembrane C-terminal region (amino acids 505-571) of rat NHE1 in the same region that binds CHP1 and CHP2. The interaction was further validated by coimmunoprecipitation and coimmunolocalization experiments using full-length CHP3 and wild-type NHE1 in transfected Chinese hamster ovary AP-1 cells. Simultaneous mutation of four hydrophobic residues within this region ((530)FLDHLL(535)) to either Ala, Gln, or Arg (FL-A, FL-Q, or FL-R) abrogated this interaction both in vitro and in intact cells. The NHE1 mutants were sorted properly to the cell surface but showed markedly reduced (FL-A) or minimal (FL-R and FL-Q) activity. Interestingly, and contrary to an earlier finding, ectopic coexpression of CHP3 up-regulated the cell surface activity of wild-type NHE1. This stimulation was not observed with the CHP3 binding-defective mutants. Mechanistically, overexpression of CHP3 did not alter the H(+) sensitivity of wild-type NHE1 but rather promoted its biosynthetic maturation and half-life at the cell surface, thereby increasing the steady-state abundance of functional NHE1 protein.
authors.affiliation: Department of Physiology, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: Hans C
authors.initials: HC
authors.lastname: Zaun
authors.forename: Alvin
authors.initials: A
authors.lastname: Shrier
authors.forename: John
authors.initials: J
authors.lastname: Orlowski
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis; D006207:Half-Life; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011522:Protons; D051381:Rats; D012964:Sodium / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis* / Q000737:chemistry; D013816:Thermodynamics; D014162:Transfection
chemical_list: D002135:Calcium-Binding Proteins; D050505:Mutant Proteins; D020033:Protein Isoforms; D011522:Protons; C452850:Slc9a1 protein, rat; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; C486009:TESC protein, human; D012964:Sodium