PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells

Elevated levels of intracellular Ca²⁺ ([Ca²⁺]i) inhibit Na⁺/H⁺ exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC- directly binds NHE3, an interaction that is necessary for [Ca²⁺]i inhibition of NHE3 activity, and that PLC- γ Src homology 2 (SH2) domains may scaffold Ca²⁺ signaling proteins necessary for regulation of NHE3 activity. [Ca²⁺]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 [Ca²⁺]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 Y416 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 [Ca²⁺]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 [Ca²⁺]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca²⁺]i conditions, PLC- γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC- γ from NHE3 and subsequent endocytosis of NHE3.