Phospholipase C-γ Binds Directly to the Na+/H+ Exchanger 3 and Is Required for Calcium Regulation of Exchange Activity

Abstract

Multiple studies suggest that phospholipase C-␥ (PLC-␥) contributes to regulation of sodium/hydrogen exchanger 3 (NHE3) in the small intestine, although the mechanism(s) for this regulation remain unknown. We demonstrate here that PLC-␥ binds directly to the C terminus of NHE3 and exists in similar sized multiprotein complexes as NHE3. This binding is dynamic and decreases with elevated [Ca 2؉ ] i . The PLC-␥-binding site in NHE3 was identified (amino acids 586 -605) and shown to be a critical regulatory domain for protein complex formation, because when it is mutated, NHE3 binding to PLC-␥ as well as NHERF2 is lost. An inhibitory peptide, which binds to the Src homology 2 domains contained in PLC-␥ without interrupting binding of PLC-␥ to NHE3, was used to probe a non-lipase-dependent role of PLC-␥. In the presence of this peptide, carbachol-stimulated calcium inhibition of NHE3 was lost. These results mirror previous studies with the transient receptor potential channel and suggest that PLC-␥ may play a common role in regulating the cell-surface expression of ion transporters.

NHERF1 and NHERF2 are necessary for multiple but usually separate aspects of basal and acute regulation of NHE3 activity

“…Elevation of free intracellular Ca 2ϩ by ionophore (4-bromo-A23187) or induced by carbachol (M3 cholinergic receptor mediated) inhibits NHE3 activity (9,17,24,26,28,52,54). Previous studies have shown that NHE3 inhibition by elevated Ca 2ϩ in PS120 cells requires NHERF2 but not NHERF1 (17).…”

Oxidative Stress Causes Renal Angiotensin II Type 1 Receptor Upregulation, Na + /H + Exchanger 3 Overstimulation, and Hypertension

“…Also, Ca 2ϩ via PLC-␥ has been shown to inhibit NHE3 in fibroblasts. 38 These discrepancies could be either because of a difference in the cell system used or in response to various hormones used to increase Ca 2ϩ signaling and/or isoformspecific PLC activation. Our data suggest that, in proximal tubules, Ang II increases intracellular Ca 2ϩ , causing tyrosine phosphorylation/activation of Jak2, which, in turn, phosphorylates CaM.…”

ANG II, ANG-(1-7), ALDO and AVP biphasic effects on Na+/H+ transport: the role of cellular calcium

“…The more consistent characteristics of NHE3 include the following: 1) it is activated under basal conditions, 2) under basal conditions, NHE3 interacts with Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), 3) only the γ isoform of this kinase associates with NHE3, 4) the NHE3 C-terminal domain, which is necessary for CaMKII binding under basal conditions, is 586-605 aa long and was predicted using multiple modeling programs to be α-helical, and 5) binding is rapidly reduced under conditions involving elevated physiological levels of Ca 2+ [13]. However, other proteins associate with NHE3, including NHERF1-4, phospholipase Cγ, and CK2α [43,44]. In addition, the CaMKII-mediated inhibition of basal NHE3 activity is NHERF2-dependent, occurs as a result of changes in the NHE3 turnover number and is associated with the phosphorylation of NHE3.…”

“…This binding is rapidly reduced by a discrete increase in [Ca2+]i, which causes a conformational change in CaMKII that allows high-affinity interactions with target proteins and prevents the inactivation of the kinase by re-association between the catalytic domain and the autoinhibitory domain after Ca 2+ returns to basal levels [13,47,48]. Other proteins that associate with NHE3 require this NHE3 domain, and these include NHERF1-4, phospholipase Cγ and CK2α [43,44]. The CaMKII-mediated inhibition of basal NHE3 activity is NHERF2-dependent, modifies NHE3 turnover rates, and causes the phosphorylation of NHE3.…”