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Originally published In Press as doi:10.1074/jbc.M206629200 on August 29, 2002

J. Biol. Chem., Vol. 277, Issue 45, 42623-42632, November 8, 2002
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Clathrin-mediated Endocytosis and Recycling of the Neuron-specific Na+/H+ Exchanger NHE5 Isoform
REGULATION BY PHOSPHATIDYLINOSITOL 3'-KINASE AND THE ACTIN CYTOSKELETON*

Katalin SzásziDagger §, Anders PaulsenDagger , Elöd Z. Szabó||, Masayuki Numata||, Sergio GrinsteinDagger **, and John Orlowski||Dagger Dagger

From the || Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada and the Dagger  Programme in Cell Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada

Mammalian Na+/H+ exchangers (NHEs) are a family of integral membrane proteins that play central roles in sodium, acid-base, and cell volume homeostasis. The recently cloned NHE5 isoform is expressed predominantly in brain, but its functional and cellular properties are poorly understood. To facilitate its characterization, an epitope-tagged construct of NHE5 was ectopically expressed in nonneuronal and neuronal cells. In NHE-deficient Chinese hamster ovary AP-1 cells, NHE5 localized at the plasmalemma, but a significant fraction accumulated intracellularly in vesicles that concentrated in a juxtanuclear region. Similarly, in nerve growth factor-differentiated neuroendocrine PC12 cells and primary hippocampal neurons, immunolabeling of NHE5 was detected in endomembrane vesicles in the perinuclear region of the cell body but also along the processes. More detailed characterization in AP-1 cells using organelle-specific markers showed that NHE5 co-localized with internalized transferrin, a marker of recycling endosomes. Transient transfection of a dominant negative mutant of dynamin-1, which inhibits clathrin-mediated endocytosis, blocked uptake of transferrin as well as internalization of NHE5. Likewise, wortmannin inhibition of phosphatidylinositol 3'-kinase, a lipid kinase implicated in endosomal traffic, induced coalescence of vesicles containing NHE5 and caused a pronounced inhibition of plasmalemmal Na+/H+ exchange. By contrast, disruption of the F-actin cytoskeleton with cytochalasin D increased cell surface NHE5 activity and abundance. These observations demonstrate that NHE5 is localized to the recycling endosomal pathway and is dynamically regulated by phosphatidylinositol 3'-kinase and by the state of F-actin assembly.

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

§ Supported by a CIHR fellowship.

These authors contributed equally to this work.

** Cross-appointed to the Department of Biochemistry of the University of Toronto. Supported by a Distinguished Investigator Award from the CIHR and the current holder of the Pitblado Chair in Cell Biology.

Dagger Dagger Supported by an Investigator Award from the CIHR. 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@mcgill.ca.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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