PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class I PDZ-binding motifs

Rafiquel I Sarker,Chung-Ming Tse,Mark Donowitz,Jianbo Yang,Boyoung Cha,Tian-E Chen,Molee Chakraborty,Nicholas C Zachos,Varsha Singh

doi:10.1074/jbc.m116.774489

Rafiquel I Sarker, Chung-Ming Tse + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m116.774489

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Abstract

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.

Highlights

To whom correspondence should be addressedII (X⌽X⌽-COOH), and Class III (X(D/E)X⌽-COOH) where ⌽ represents any hydrophobic amino acid residue and X is any residue [5,6,7,8,9]
NHE3 directly binds Na؉/H؉ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation
GST-NHERF1 or GST-NHERF2 protein immobilized on glutathione-Sepharose௡ beads was used for “pulldown” assays with 35S-labeled His6-tagged NHE3 C-terminal truncations (C refers to C terminus) C589, C610, C660, C689, C711, and C832

Summary

To whom correspondence should be addressed

II (X⌽X⌽-COOH), and Class III (X(D/E)X⌽-COOH) where ⌽ represents any hydrophobic amino acid residue and X is any residue [5,6,7,8,9]. Regulation of NHE3 activity involves two identified multiprotein complexes that form on the NHE3 intracellular regulatory domain. These complexes are highly dynamic as part of this regulation, and many of the proteins involved bind the NHERF scaffolds. NHE3 interacts with NHERF2 via an internal sequence (aa 586 – 660) within the cytoplasmic domain of NHE3, but binding to the C-terminal PDZ domain-binding motif (PBM) that is present in NHE3 was not observed [16]. In attempting to resolve these differences, we identified an internal PDZ domain-binding site in the NHE3 C terminus as a Class II PDZ domain-binding motif (-595CLDM-) and have characterized the functional consequences of mutating both NHE3 PBMs

Results

Discussion

X Increased X Decreaseda

Experimental procedures