NHERF2/NHERF3 Hetero-dimerization and Macro-Complex Formation are Required for the Inhibition of NHE3 Activity by Carbachol [Signal Transduction]

May 27th, 2014 by Yang, J., Singh, V., Chen, T.-E., Sarker, R., Xiong, L., Cha, B., Jin, S., Li, X., Tse, C. M., Zachos, N. C., Donowitz, M.

NHERF1, NHERF2 and NHERF3 belong to the NHERF (Na+/H+ exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins. Individually, each NHERF protein was shown involved in the regulation of multiple receptors or transporters including Na+/H+ exchanger 3 (NHE3). Though NHERF dimerizations have been reported, results have been inconsistent and the physiological function of NHERF dimerizations is still unknown. The current study semi-quantitatively compared the interaction strength among all possible homo-dimerizations and hetero-dimerizations of these three NHERF proteins by pull-down and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 hetero-dimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 hetero-dimerization was confirmed by FRET and FRAP. NHERF2/NHERF3 hetero-dimerization is mediated by PDZ domains of NHERF2 and the carboxyl terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in hetero-dimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 hetero-dimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4 and PKCα, promoting formation of NHE3 macro-complexes. This study suggests that NHERF2/NHERF3 hetero-dimerization mediates the formation of NHE3 macro-complexes, which are required for the inhibition of NHE3 activity by carbachol.
  • Posted in Journal of Biological Chemistry, Publications
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