The NHE3 Juxtamembrane Cytoplasmic Domain Directly Binds Ezrin: Dual Role in NHE3 Trafficking and Mobility in the Brush Border

Abstract

Based on physiological studies, the epithelial brush-border (BB) Na+/H+ antiporter3 (NHE3) seems to associate with the actin cytoskeleton both by binding to and independently of the PDZ domain containing proteins NHERF1 and NHERF2. We now show that NHE3 directly binds ezrin at a site in its C terminus between aa 475-589, which is separate from the PSD95/dlg/zonular occludens-1 (PDZ) interacting domain. This is an area predicted to be alpha-helical, with a positive aa cluster on one side (K516, R520, and R527). Point mutations of these positively charged aa reduced (NHE3 double mutant [R520F, R527F]) or abolished (NHE3 triple mutant [K516Q, R520F, R 527F]) ezrin binding. Functional consequences of these NHE3 point mutants included the following. 1) A marked decrease in surface amount with a greater decrease in NHE3 activity. 2) Decreased surface expression due to decreased rates of exocytosis and plasma membrane delivery of newly synthesized NHE3, with normal total expression levels and slightly reduced endocytosis rates. 3) A longer plasma membrane half-life of mutant NHE3 with normal total half-life. 4) Decreased BB mobile fraction of NHE3 double mutant. These results show that NHE3 binds ezrin directly as well as indirectly and suggest that the former is related to 1) the exocytic trafficking of and plasma membrane delivery of newly synthesized NHE3, which determines the amount of plasma membrane NHE3 and partially determines NHE3 activity, and 2) BB mobility of NHE3, which may increase its delivery from microvilli to the intervillus clefts, perhaps for NHE3-regulated endocytosis.

Dynamic Na+-H+ Exchanger Regulatory Factor-1 Association and Dissociation Regulate Parathyroid Hormone Receptor Trafficking at Membrane Microdomains

“…It has been proposed that a ternary complex comprised of the PTHR, NHERF1, and ezrin is formed by the interaction of the FERM domain of ezrin with the intracellular carboxyl-terminal tail of the PTHR (40). The sodium-hydrogen exchanger-3 (NHE-3), which like the PTHR possesses an atypical PDZ recognition motif, STHM, also binds directly to ezrin (50). Furthermore, NHE-3 binds both to NHERF1 and ezrin to form a ternary complex (51).…”

Casein Kinase 2 Binds to the C Terminus of Na+/H+exchanger 3 (NHE3) and Stimulates NHE3 Basal Activity by Phosphorylating a Separate Site in NHE3

“…TBB and DMAT both caused concentration-dependent inhibition of NHE3 activity with different effects in NHE-WT and the NHE3-S719A mutant. DMAT, a specific inhibitor of CK2 (Obenauer et al, 2003;Cha et al, 2006), inhibited NHE3 wild-type activity by 55% at 30 M ( Figure 4A). TBB, a potent inhibitor of both CK2 and CK1 (it inhibits CK1 at higher concentrations) (Sarno et al, 2002;Ruzzene et al, 2002) exerted a maximum of 85% inhibition of wild-type NHE3 activity at 30 M (similar effect at 100 M) and caused 50% inhibition at 3-5 M TBB ( Figure 4B).…”

“…The 6x His-tagged fusion proteins made in the pET30a vector included four fragments of the NHE3 C terminus: F1 (amino acids [aa] 475-589), F2 (aa 590-667), F3 (aa 668-747), and F4 (aa 748-832) (Cha et al, 2006). His 6 -tagged fusion proteins were expressed in Escherichia coli Rosetta 2 (DHE-3) cells, and crude cell extract was prepared following the Invitrogen protocol under native conditions.…”

“…NHS-SS-biotin was used to determine the percentage of total NHE3 on the plasma membrane; rates of exocytosis and endocytosis; and with 35 S labeling, delivery rates of newly synthesized NHE3 to the plasma membrane, as described previously (Cha et al, 2006). PS120 cells stably expressing HA-NHE3-WT and HA-NHE3-S719A were grown to 70 -80% confluence in 10-cm Petri dishes.…”

“…To measure exocytic insertion of NHE3 (also called endocytic recycling), N-hydroxysuccinimide (NHS)-reactive sites on proteins on the PS120 cell surface were first blocked by pretreatment with sulfo-NHS-acetate with some slight modifications (Cha et al, 2006). Cells were rinsed twice with PBS-Ca-Mg (PBS with 0.1 mM Ca 2ϩ and 1 mM Mg 2ϩ ) at 4°C.…”