BetaPix up-regulates Na+/H+ exchanger 3 through a Shank2-mediated protein-protein interaction.
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.
Lee
Jung-Soo
JS
Department of Pharmacology and Brain Korea 21 Project for Medical Science, Institute of Gastroenterology, Yonsei University College of Medicine, Seoul 120-752, Korea.
Lee
Young Mee
YM
Kim
Joo Young
JY
Park
Hyun Woo
HW
Grinstein
Sergio
S
Orlowski
John
J
Kim
Eunjoon
E
Kim
Kyung Hwan
KH
Lee
Min Goo
MG
D000818:Animals; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011522:Protons; D051381:Rats; D017207:Rats, Sprague-Dawley; D064067:Rho Guanine Nucleotide Exchange Factors; D012964:Sodium / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011522:Protons; D064067:Rho Guanine Nucleotide Exchange Factors; C122162:SHANK2 protein, human; C000613385:SLC9A3 protein, human; C485686:Shank2 protein, rat; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D020741:rho GTP-Binding Proteins