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

Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na(+)/H(+) exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na(+) absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca(2+) and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca(2+)-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca(2+)-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent.