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 se quences in its C-terminal region that resemble SH3 (Src homology 3)-binding domains. We investigated whether these regions ((PPS)-P-743-VTPAP(750), te rmed Pro-1, and (VPPKPPP792)-V-786, termed Pro-2) can bind to SH3 domains a nd whether they are essential for NHE2 function and targeting. A fusion pro tein containing the Pro-1 region showed promiscuous binding to SH3 domains of several proteins in vitro, whereas a Pro-2 fusion bound preferentially t o domains derived from kinases. In contrast, cytoplasmic regions of NHE1, N HE3, or NHE4 failed to interact. When expressed in antiporter-deficient cel ls, truncated NHE2 lacking both Pro-rich regions catalyzed Na+/H+ exchange, retained sensitivity to intracellular ATP, and was activated by hyperosmol arity, resembling full-length NHE2, The role of the Pro-rich regions in sub cellular 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 cont rast, a mutant devoid of both Pro-1 and Pro-2 was preferentially sorted to the basolateral surface but also accumulated intracellularly. These observa tions indicate that the region encompassing Pro-1 is essential for appropri ate subcellular targeting of NHE2.