Abstract
Na(+)/H(+) exchanger 3 (NHE3) plays an important role in neutral Na(+) transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of betaPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and betaPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, betaPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of betaPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in betaPix-mediated NHE3 regulation. Knockdown of endogenous betaPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that betaPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.
Highlights
It has been shown that adaptor proteins with PDZ (PSD-95/ discs large/ZO-1) domains play an important role in the membrane expression and acute regulation of Na؉/H؉ exchanger 3 (NHE3) activity in polarized epithelia
Shank2 has been shown to be localized to the apical poles of pancreatic, colonic, hemagglutinin; GFP, green fluorescent protein; BCECF, acetoxymethyl ester of 2Ј,7Ј-bis(carboxyethyl)-5(6)-carboxyfluorescein; TxB, toxin B; DSP, dithiobis(succinimidyl propionate); CMV, cytomegalovirus; Madin-Darby canine kidney (MDCK), MadinDarby canine kidney cell; small interfering RNA (siRNA), small interference RNA; GST, glutathione S-transferase; HEK, human embryonic kidney; pHi, intracellular pH; CFTR, cystic fibrosis transmembrane conductance regulator
We describe a novel mechanism of NHE3 regulation by Pix
Summary
It has been shown that adaptor proteins with PDZ (PSD-95/ discs large/ZO-1) domains play an important role in the membrane expression and acute regulation of NHE3 activity in polarized epithelia. Shank2 associates with NHE3 and up-regulates the membrane expression and basal activity of NHE3 in epithelial cells [5]. It is conceivable that Pix associates with the Shank2-NHE3 complex and participates in NHE3 regulation by activating Rho GTPases near the apical pole in epithelial cells.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
