NDLI: Identification of Cytoplasmic Domains within the Epithelial Na+ Channel Reactive at the Plasma Membrane

Content Provider	Journal of Biological Chemistry (JBC)
Author	Hendron, Eunan Patel, Pravina Hausenfluke, Melinda Gamper, Nikita Shapiro, Mark S. Booth, Rachell E. Stockand, James D.
Abstract	The activity of membrane proteins is controlled, in part, by protein-protein interactions localized to the plasma membrane. In the current study, domains within the epithelial Na+ channel (ENaC) reactive at the plasma membrane were identified using a novel yeast one-hybrid screen. The cytosolic N terminus of αENaC and the cytosolic C termini of α-, β-, and γENaC contained domains reactive at the plasma membrane. Fluorescent micrographs of epithelial cells overexpressing fusion proteins of enhanced green fluorescent protein and mENaC cytosolic domains were consistent with those in yeast. A novel membrane reactive domain within the cytosolic C terminus of γ-mENaC was localized to the 17 amino acids between residues Thr584–Pro600. Two overlapping internalization signals within the C terminus of γ-mENaC, a WW-binding domain (PY motif) and a tyrosine-based endocytic signal, were additive with respect to decreasing complementation and expression levels of hybrid proteins. Decreases in expression levels of hybrid proteins containing the PY and endocytic motif were reversed with latrunculin A, an inhibitor of endosomal movement. Decreases in complementation and expression levels of hybrid proteins mediated by the combined PY and overlapping endocytic motif proceeded in the absence of established ubiquitination sites within ENaC. In addition, the endocytic motif was active in the absence of the PY motif, demonstrating that these two domains, while possibly interacting, also have discrete functions. The novel domains within the cytosolic N terminus of αENaC and the C termini of α-, β-, and γENaC identified here are likely to be involved in protein-protein and/or protein-lipid interactions localized to the plasma membrane. We hypothesize that these newly identified domains play a role in modulating ENaC activity.
Related Links	http://www.jbc.org/content/277/37/34480.abstract
Ending Page	34488
Starting Page	34480
Page Count	9
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	37
Volume Number	277
DOI	10.1074/jbc.M204615200
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2002-09-13
Access Restriction	Open
Subject Keyword	Epithelial Na+ channel (ENaC) Chinese hamster ovary (CHO) Dulbecco's modified Eagle's medium (DMEM) Enhanced green fluorescent protein (EGFP) Tris-buffered saline (TBS) Human Son-of-Sevenless (hSOS) MEMBRANE TRANSPORT STRUCTURE FUNCTION AND BIOGENESIS
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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