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J. Biol. Chem., Vol. 283, Issue 18, 12456-12467, May 2, 2008

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Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na⁺/H⁺ Exchanger NHE1 Isoform^*

From the Department of Physiology, McGill University, Montréal, Québec H3G 1Y6, Canada

Calcineurin B homologous protein (CHP) 1 and 2 are Ca²⁺-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 (⁵³⁰FLDHLL⁵³⁵) 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.

Received for publication, January 10, 2008 , and in revised form, February 28, 2008.

^* This work was supported by grants from the Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Physiology, McGill University, McIntyre Medical Science Bldg., 3655 Promenade Sir-William-Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-8335; Fax: 514-398-7452; E-mail: john.orlowski{at}mcgill.ca.

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