Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5*
- Departments of ‡Biochemistry and Molecular Biology and §Cellular and Physiological Sciences, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
- 2 An MSFHR scholar. To whom correspondence should be addressed: Tel.: 604-822-7728; Fax: 604-822-5227; E-mail: mnumata{at}interchange.ubc.ca.
Abstract
NHE5 is a brain-enriched Na+/H+ exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.
Footnotes
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↵3 The abbreviations used are: NHE, Na+/H+ exchanger; SCAMP, secretory carrier membrane protein; EH, Eps15 homology; GFP, green fluorescent protein; EGFP, enhanced GFP; PBS, phosphate-buffered saline; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; BCECF/AM, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester.
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↵* This work was supported in part by Canadian Institutes of Health Research Operating Grants MOP144919 (to M. N.) and MOP77616 (to J. C.), and by Michael Smith Foundation for Health Research (MSFHR) establishment grants (to M. N.).
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↵1 Recipient of an MSFHR junior graduate studentship and Natural Sciences and Engineering Research Council of Canada CGS-M and CGS-D scholarships.
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- Received September 11, 2008.
- Revision received February 2, 2009.
- The American Society for Biochemistry and Molecular Biology, Inc.
