Proline-rich motifs of the Na+/H+ exchanger 2 isoform. Binding of Src homology domain 3 and role in apical targeting in epithelia.
The NHE2 isoform of the Na+/H+ exchanger (NHE) displays two proline-rich sequences in its C-terminal region that resemble SH3 (Src homology 3)-binding domains. We investigated whether these regions (743PPSVTPAP750, termed Pro-1, and 786VPPKPPP792, termed Pro-2) can bind to SH3 domains and whether they are essential for NHE2 function and targeting. A fusion protein containing the Pro-1 region showed promiscuous binding to SH3 domains of several proteins in vitro, whereas a Pro-2 fusion bound preferentially to domains derived from kinases. In contrast, cytoplasmic regions of NHE1, NHE3, or NHE4 failed to interact. When expressed in antiporter-deficient cells, truncated NHE2 lacking both Pro-rich regions catalyzed Na+/H+ exchange, retained sensitivity to intracellular ATP, and was activated by hyperosmolarity, resembling full-length NHE2. The role of the Pro-rich regions in subcellular targeting was examined by transfection of epitope-tagged forms of NHE2 in porcine renal epithelial LLC-PK1 cells. Both full-length and Pro-2-truncated NHE2 localized almost exclusively to the apical membrane. By contrast, a mutant devoid of both Pro-1 and Pro-2 was preferentially sorted to the basolateral surface but also accumulated intracellularly. These observations indicate that the region encompassing Pro-1 is essential for appropriate subcellular targeting of NHE2.
Chow
C W
CW
Cell Biology Programme, Hospital for Sick Children, University of Toronto, Ontario M5G 1X8, Canada.
Woodside
M
M
Demaurex
N
N
Yu
F H
FH
Plant
P
P
Rotin
D
D
Grinstein
S
S
Orlowski
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D006224:Cricetinae; D004847:Epithelial Cells / Q000737:chemistry; D009994:Osmolar Concentration; D011392:Proline / Q000032:analysis*; D011994:Recombinant Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics; D014162:Transfection; D018909:src Homology Domains
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D000255:Adenosine Triphosphate; D011392:Proline