title: PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells.
abstract: Elevated levels of intracellular Ca(2+) ([Ca(2+)]i) inhibit Na(+)/H(+) exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca(2+)]i inhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca(2+) signaling proteins necessary for regulation of NHE3 activity. [Ca(2+)]i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca(2+)]i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y(416) phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca(2+)]i inhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca(2+)]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca(2+)]i conditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.
authors.affiliation: Department of Medicine/Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. nzachos1@jhmi.edu
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Zachos
authors.forename: Luke J
authors.initials: LJ
authors.lastname: Lee
authors.forename: Olga
authors.initials: O
authors.lastname: Kovbasnjuk
authors.forename: Xuhang
authors.initials: X
authors.lastname: Li
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
mesh_terms: D018712:Analgesics, Non-Narcotic / Q000494:pharmacology*; D018938:Caco-2 Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D016391:Genes, src; D006801:Humans; D051966:Phospholipase C gamma / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*
chemical_list: C412373:AG 1879; D018712:Analgesics, Non-Narcotic; D011743:Pyrimidines; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D002217:Carbachol; D016392:Proto-Oncogene Proteins pp60(c-src); D051966:Phospholipase C gamma; D002118:Calcium
keywords: NHE3; PLC-γ; c-Src; complexes