The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.
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.
Affiliation: Departments of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.Show MeSH
Mentions: For Figures 2–5 and 8, 1 nmol of recombinant GST-N–ezrin was used as bait for pull-downs. For binary interaction studies, 3 nmol of purified proteins were used as prey. For competition experiments between two NHERF fragments or full-length proteins, 3 nmol of each purified protein were used. For competition experiments described in Figure 4, 1 nmol of GST-N–ezrin was first pre-incubated with 3 nmol of His6–thioredoxin or His6–thioredoxin fused NHERF C-terminus fragments for 30 min, then 1 mg of Caco-2 cell lysate was added. The volume of final mixture was adjusted to 500 μl. Each bait–prey mixture was mixed with 10 μl of pre-washed GSH-resin and incubated at 4°C for 3 h on a rotating shaker. Resin was washed four times and eluted with lysis buffer plus 10 mM glutathione. For Figure 9, 3 μg of purified GST-N–ezrin was mixed with 1.5 mg of HEK-293A cell lysate expressing FLAG–NHERF2 or mutants and 10 μl of GSH–resin for overnight incubation at 4°C. Resin was washed four times and finally eluted with 2× Laemmli buffer at 80°C.
Affiliation: Departments of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.