A C-terminal PDZ motif in NHE3 binds NHERF-1 and enhances cAMP inhibition of sodium-hydrogen exchange.
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.
Weinman
Edward J
EJ
Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Wang
Yu
Y
Wang
Fengying
F
Greer
Charmaine
C
Steplock
Deborah
D
Shenolikar
Shirish
S
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D011817:Rabbits; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D010750:Phosphoproteins; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D000242:Cyclic AMP