NHERF2/NHERF3 Protein Heterodimerization and Macrocomplex Formation Are Required for the Inhibition of NHE3 Activity by Carbachol*
- Jianbo Yang‡,
- Varsha Singh‡,
- Tian-E Chen‡,
- Rafiquel Sarker‡,
- Lishou Xiong‡1,
- Boyoung Cha‡,
- Shi Jin‡,
- Xuhang Li‡,
- C. Ming Tse‡,
- Nicholas C. Zachos‡ and
- Mark Donowitz‡,§2
- From the ‡Department of Medicine, Division of Gastroenterology and
- §Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
- ↵2 To whom correspondence should be addressed: Division of Gastroenterology and Hepatology, Dept. of Medicine, 925 Ross Research Bldg., Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205-2195. Tel.: 410-955-9675; Fax: 410-955-9677; E-mail: mdonowit{at}jhmi.edu.
Abstract
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 has been shown to be involved in the regulation of multiple receptors or transporters including Na+/H+ exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization 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 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.
- Epithelial Cell
- Fluorescence Resonance Energy Transfer (FRET)
- Protein-Protein Interaction
- Scaffold Protein
- Trafficking
- Dimerization
- Macrocomplex
- Microvillus
- NHE3 (Na+/H+ Exchanger 3)
- NHERF Proteins
Footnotes
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↵* This work was supported, in whole or in part, by National Institutes of Health Grants R01DK26523, R01DK61765, P01DK072084, K08DK088950, and P30 DK089502 (The NIDDK Hopkins Digestive Diseases Basic and Translational Research Core Center).
- Received March 2, 2014.
- Revision received May 18, 2014.
- © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
