Ca²⁺-dependent inhibition of NHE3 requires PKCα which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes

Abstract

The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKCalpha pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKCalpha from cytosol to membrane. PKCalpha bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKCalpha and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKCalpha is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.

P2Y 1 receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2

“…NHERF family proteins are known to associate with many of the intermediates involved in G␣ q -mediated signaling, including G␣ q itself (24), PKC␣ (25), and PLC␤ (26). Thus, NHERF-2 may control P2Y 1 R activity by acting as a scaffold to link these key signaling proteins in the vicinity of the receptor, allowing for a more sustained signaling response.…”

“…P2Y 1 R exerts effects on cellular physiology mainly through coupling to G␣ q. Interestingly, NHERF family proteins are known to interact with a number of proteins implicated in G␣ q -mediated signal transduction, including G␣ q itself (24), PKC␣ (25), and subtypes of PLC␤ (26). NHERF-2 might therefore be able to strongly influence P2Y 1 R-mediated signaling efficacy by clustering these key signaling components in the vicinity of the receptor.…”

“…Put together, these results reveal that expression of NHERF-2 in C6 glioma cells greatly prolongs the duration of P2Y 1 R-mediated Ca 2ϩ signaling while having a slight effect on the response magnitude. Because NHERF-2 can interact with and regulate the activity of various G q signaling components (24)(25)(26), the effects of NHERF-2 on P2Y 1 R-mediated Ca 2ϩ signaling observed in the experiments with the C6 glioma cells might reflect general effects of NHERF-2 on the efficiency of G q signaling (or on Ca 2ϩ signaling in general) rather than specific effects on P2Y 1 R-mediated signaling due to the NHERF-2 interaction with P2Y 1 R. Thus, we performed parallel experiments in human 1321N1 astrocytoma cells that lack endogenous P2YRs (8) but express significant levels of endogenous NHERF-2 (data not shown). These glial cells lacking endogenous P2Y 1 R allowed for direct comparisons between WT P2Y 1 Rs and L373A mutant receptors that are unable to bind to the endogenous NHERF-2 expressed in these cells.…”

Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

“…Motif-Calcium-dependent short term regulation of NHE3 has been studied in transfected PS120 cells (19,22). Thus we followed this approach and used our previously established HEK cells stably expressing EGFP-DRA (15).…”

Lysophosphatidic acid prevents apoptosis of Caco-2 colon cancer cells via activation of mitogen-activated protein kinase and phosphorylation of Bad

“…LPA 2 has carboxyl-terminal motifs that allow interaction with a PDZ (PSD-95/DlgA/ZO-1) domain of the Na + /H + exchanger regulatory factor 2 (NHERF2), which functions as a scaffold to link signaling proteins, such as PKA and PKCα, to GPCRs and transporters at the cell surface [21,23,[29][30][31]. NHERF2 links LPA 2 to PLC-β3 and hence affects the activation of downstream targets, such as Erk and cyclooxygenase-2 [23].…”