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The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.

Yang J, Sarker R, Singh V, Sarker P, Yin J, Chen TE, Chaerkady R, Li X, Tse CM, Donowitz M - Biochem. J. (2015)

Bottom Line: The current study found that NHERF1/2 contain an ERM-binding regulatory sequence (EBRS), which facilitates the interaction between the EBD and ezrin.Furthermore, phosphorylation of Ser(303) located in the EBRS of NHERF2, decreases the binding affinity for ezrin, dislocates apical NHERF2 into the cytosol and increases the NHERF2 microvillar mobility rate.Moreover, increased phosphorylation of Ser(303) was functionally significant preventing acute stimulation of NHE3 (Na(+)-H(+) exchanger 3) activity by dexamethasone.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.

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Expression of the NHERF2–S303D mutant in OK cells does not allow the NHERF2-dependent dexamethasone stimulation of NHE3 activityNHE3 activity was measured in OK cells transfected with NHERF2–WT, S303A and S303D and exposed to dexamethasone, 10 μM, 4 h. Dexamethasone stimulated NHE3 activity equally in cells transfected with WT and NHERF2–S303A but not in cells transfected with NHERF2–S303D. Results are means ± S.E.M. of four separate experiments. P-values compare dexamethasone stimulation of NHE3 in WT NHERF2 expressing cells with that in cells expressing NHERF2–S303A and S303D (paired t tests).
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Figure 11: Expression of the NHERF2–S303D mutant in OK cells does not allow the NHERF2-dependent dexamethasone stimulation of NHE3 activityNHE3 activity was measured in OK cells transfected with NHERF2–WT, S303A and S303D and exposed to dexamethasone, 10 μM, 4 h. Dexamethasone stimulated NHE3 activity equally in cells transfected with WT and NHERF2–S303A but not in cells transfected with NHERF2–S303D. Results are means ± S.E.M. of four separate experiments. P-values compare dexamethasone stimulation of NHE3 in WT NHERF2 expressing cells with that in cells expressing NHERF2–S303A and S303D (paired t tests).

Mentions: The functional significance of phosphorylation of NHERF2–Ser303 was examined by exposing OK cells to dexamethasone for 4 h. Dexamethasone is known to stimulate NHE3 by an acute NHERF2-dependent effect that does not involve stimulation of transcription of NHE3 [43]. OK cells do not express detectable endogenous NHERF2 [43]. As shown in Figure 11, dexamethasone exposure stimulated NHE3 similarly in the presence of exogenous WT NHERF2 and NHERF2–S303A but not when NHERF2–S303D was expressed. NHE3–S303D basal activity was increased compared with WT NHE3 (Figure 11). These results are consistent with the dexamethasone effect requiring NHERF2–ezrin binding to mediate the assembly of a NHE3 signalling complex on the apical membrane [43] and demonstrate functional significance of phosphorylation of NHERF2–Ser303.


The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.

Yang J, Sarker R, Singh V, Sarker P, Yin J, Chen TE, Chaerkady R, Li X, Tse CM, Donowitz M - Biochem. J. (2015)

Expression of the NHERF2–S303D mutant in OK cells does not allow the NHERF2-dependent dexamethasone stimulation of NHE3 activityNHE3 activity was measured in OK cells transfected with NHERF2–WT, S303A and S303D and exposed to dexamethasone, 10 μM, 4 h. Dexamethasone stimulated NHE3 activity equally in cells transfected with WT and NHERF2–S303A but not in cells transfected with NHERF2–S303D. Results are means ± S.E.M. of four separate experiments. P-values compare dexamethasone stimulation of NHE3 in WT NHERF2 expressing cells with that in cells expressing NHERF2–S303A and S303D (paired t tests).
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Related In: Results  -  Collection

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Figure 11: Expression of the NHERF2–S303D mutant in OK cells does not allow the NHERF2-dependent dexamethasone stimulation of NHE3 activityNHE3 activity was measured in OK cells transfected with NHERF2–WT, S303A and S303D and exposed to dexamethasone, 10 μM, 4 h. Dexamethasone stimulated NHE3 activity equally in cells transfected with WT and NHERF2–S303A but not in cells transfected with NHERF2–S303D. Results are means ± S.E.M. of four separate experiments. P-values compare dexamethasone stimulation of NHE3 in WT NHERF2 expressing cells with that in cells expressing NHERF2–S303A and S303D (paired t tests).
Mentions: The functional significance of phosphorylation of NHERF2–Ser303 was examined by exposing OK cells to dexamethasone for 4 h. Dexamethasone is known to stimulate NHE3 by an acute NHERF2-dependent effect that does not involve stimulation of transcription of NHE3 [43]. OK cells do not express detectable endogenous NHERF2 [43]. As shown in Figure 11, dexamethasone exposure stimulated NHE3 similarly in the presence of exogenous WT NHERF2 and NHERF2–S303A but not when NHERF2–S303D was expressed. NHE3–S303D basal activity was increased compared with WT NHE3 (Figure 11). These results are consistent with the dexamethasone effect requiring NHERF2–ezrin binding to mediate the assembly of a NHE3 signalling complex on the apical membrane [43] and demonstrate functional significance of phosphorylation of NHERF2–Ser303.

Bottom Line: The current study found that NHERF1/2 contain an ERM-binding regulatory sequence (EBRS), which facilitates the interaction between the EBD and ezrin.Furthermore, phosphorylation of Ser(303) located in the EBRS of NHERF2, decreases the binding affinity for ezrin, dislocates apical NHERF2 into the cytosol and increases the NHERF2 microvillar mobility rate.Moreover, increased phosphorylation of Ser(303) was functionally significant preventing acute stimulation of NHE3 (Na(+)-H(+) exchanger 3) activity by dexamethasone.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.

Show MeSH