NHERF2/NHERF3 Protein Heterodimerization and Macrocomplex Formation Are Required for the Inhibition of NHE3 Activity by Carbachol

• Published in: The Journal of biological chemistry Vol. 289; no. 29; pp. 20039 - 20053
• Main Authors: Yang, Jianbo, Singh, Varsha, Chen, Tian-E, Sarker, Rafiquel, Xiong, Lishou, Cha, Boyoung, Jin, Shi, Li, Xuhang, Tse, C.Ming, Zachos, Nicholas C., Donowitz, Mark
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.07.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Animals; Caco-2 Cells; Carbachol - pharmacology; Cell Line; Cricetinae; Dimerization; Epithelial Cell; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Fluorescence Resonance Energy Transfer (FRET); Humans; Macrocomplex; Microvillus; Multiprotein Complexes - chemistry; Multiprotein Complexes - genetics; Multiprotein Complexes - metabolism; Mutagenesis, Site-Directed; NHE3 (Na+/H+ Exchanger 3); NHERF Proteins; PDZ Domains; Phosphoproteins - chemistry; Phosphoproteins - genetics; Phosphoproteins - metabolism; Protein Multimerization; Protein-Protein Interaction; Rabbits; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Scaffold Protein; Signal Transduction; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers - antagonists & inhibitors; Sodium-Hydrogen Exchangers - chemistry; Sodium-Hydrogen Exchangers - genetics; Sodium-Hydrogen Exchangers - metabolism; Trafficking; NHERF Proteins; Macrocomplex; NHE3 (Na+/H+ Exchanger 3); Protein-Protein Interaction; Trafficking; Scaffold Protein; Epithelial Cell; Fluorescence Resonance Energy Transfer (FRET); Microvillus; Dimerization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.562413

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. Background: Previous characterization of NHERF dimerizations is inconsistent and any physiological function is largely unknown. Results: NHERF2/NHERF3 heterodimerization is the strongest NHERF dimerization and is necessary for carbachol inhibition of NHE3. Conclusion: NHERF2/NHERF3 heterodimerization brings PDZ domains together to assemble macrocomplexes for NHE3 regulation. Significance: Macrocomplexes mediated by NHERF dimerizations could increase the complicity of their regulation of NHE3 and other proteins.