A C-terminal PDZ motif in NHE3 binds NHERF-1 and enhances cAMP inhibition of sodium-hydrogen exchange.

Published

Journal Article

NHERF-1, a protein adapter containing two tandem PDZ domains, was first identified as an essential cofactor required for the phosphorylation and downregulation of NHE3 activity in response to elevated intracellular cAMP. NHERF-1 contains multiple protein interaction domains, but the mechanism by which it binds NHE3 remains unknown. Yeast two-hybrid analyses demonstrated that the C-terminal sequence, STHM, of NHE3 constitutes a PDZ motif critical for its association with NHERF-1. In this assay, NHE3 bound both PDZ-I and PDZ-II when presented as isolated domains, but mutations of the individual PDZ domains in the full-length NHERF-1 suggested a significant preference of NHE3 for the PDZ-II domain. To investigate NHERF-1/NHE3 association in cells, NHERF-1 complexes were isolated from PS120 cells expressing hexahistidine-tagged NHERF-1 and NHE3 using nickel-NTA-agarose. In these experiments, mutating the C-terminal PDZ motif still allowed NHE3 binding to NHERF-1, suggesting the presence of additional mechanisms or components that stabilized a cellular NHE3/NHERF-1 complex. Transport assays in PS120 cells, however, showed that the C-terminal PDZ motif in NHE3 and a functional PDZ-II domain in NHERF-1 were required for maximal inhibition of sodium-hydrogen exchange in response to forskolin and 8-Br-cAMP. Together, the data suggested that the PDZ interaction between the NHE3 C-terminus and a NHERF-1 PDZ domain enhanced the regulation of sodium-hydrogen exchange by cAMP-elevating hormones.

Full Text

Duke Authors

Cited Authors

  • Weinman, EJ; Wang, Y; Wang, F; Greer, C; Steplock, D; Shenolikar, S

Published Date

  • November 4, 2003

Published In

Volume / Issue

  • 42 / 43

Start / End Page

  • 12662 - 12668

PubMed ID

  • 14580213

Pubmed Central ID

  • 14580213

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi035244l

Language

  • eng

Conference Location

  • United States