Department of Medicine/Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Am J Physiol Cell Physiol. 2013 Aug 1;305(3):C266-75. doi: 10.1152/ajpcell.00277.2012. Epub 2013 May 22.
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.
细胞内钙离子浓度 ([Ca(2+)]i) 升高可抑制完整肠道中钠/氢交换器 3 (NHE3) 的活性。我们之前的研究表明，PLC-γ 可直接与 NHE3 结合，这种相互作用是 [Ca(2+)]i 抑制 NHE3 活性所必需的，而 PLC-γ Src 同源结构域 2 (SH2) 结构域可能构成调节 NHE3 活性所必需的 Ca(2+) 信号蛋白。[Ca(2+)]i 对 NHE3 活性的调节也依赖于 c-Src；然而，c-Src 参与的机制尚不清楚。我们假设 PLC-γ 的 SH2 结构域可能将 c-Src 与含有 NHE3 的复合物连接起来，从而介导 [Ca(2+)]i 对 NHE3 活性的抑制。在 Caco-2/BBe 细胞中，卡巴胆碱 (CCh) 使 NHE3 活性降低约 40%，而 c-Src 抑制剂 PP2 则可消除这种作用。CCh 处理可使 Y(416) 磷酸化，从而使 c-Src 的活性增加，最早在 1 分钟即可出现这种作用。共免疫沉淀表明，c-Src 在基础条件下与 PLC-γ 而不是 NHE3 结合，这种相互作用在 CCh 处理后迅速增加，并发生在 CCh 处理 10 分钟后 PLC-γ 和 NHE3 分离之前。最后，只有通过 PLC-γ SH2 结构域才能与 c-Src 直接结合，而通过阻断 PLC-γ SH2 结构域可防止这种相互作用。本研究表明，c-Src：1）活性是 [Ca(2+)]i 抑制 NHE3 活性所必需的；2）在 CCh 处理后迅速（约 1 分钟）激活；3）在升高的 [Ca(2+)]i 条件下，直接与 PLC-γ SH2 结构域结合，并与 PLC-γ 动态结合；4）不直接与 NHE3 结合。在升高的 [Ca(2+)]i 条件下，PLC-γ 在 PLC-γ 与 NHE3 分离和随后的 NHE3 内吞作用之前，将 c-Src 支架成含有 NHE3 的多蛋白复合物。
