NHE3 is a brush border (BB) Na⁺/H⁺ antiporter which accounts for the majority of physiologic small intestinal and renal Na+ absorption. It is regulated physiologically and in disease via changes in endocytosis/exocytosis. Paradoxically, NHE3 is fixed to the microvillar (MV) actin cytoskeleton and has little basal mobility.. This fixation requires NHE3 binding to the multi-PDZ domain scaffold proteins NHERF1 and NHERF2 and to ezrin. Coordinated release of NHE3 from the MV cytoskeleton has been demonstrated during both stimulation and inhibition of NHE3. However, the signaling molecules involved in coordinating NHE3 trafficking and cytoskeletal association have not been identified. This question was addressed by studying LPA stimulation of NHE3 in polarized renal proximal tubule OK cells which occurs via apical LPA5 receptors and is NHERF2 dependent and mediated by EGFR, Rho/ROCK and ERK. NHE3 activity was determined by BCECF/fluorometry and NHE3 microvillar mobility by FRAP/confocal microscopy using NHE3-EGFP. Apical LPA (3µM)/LPA5R stimulated NHE3 activity, increased NHE3 mobility, and decreased the NHE3/NHERF2 association. The LPA stimulation of NHE3 was also PKC dependent. PKC was necessary for LPA stimulation of NHE3 mobility and NHE3/NHERF2 association. Moreover, the LPA induced translocation to the membrane of PKC was both ERK and phospholipase C dependent with ERK acting upstream of PLC. We conclude that LPA stimulation of NHE3 exocytosis includes a signaling pathway that regulates fixation of NHE3 to the MV cytoskeleton. This involves a signaling module consisting of ERK-PLC-PKCwhich dynamically and reversibly releases NHE3 from NHERF2 to contribute to the changes in NHE3 MV mobility