Abstract
Cyclic AMP is a major second messenger that inhibits the brush border Na+/H+ exchanger NHE3. We have previously shown that either of two related regulatory proteins, E3KARP or NHERF, is necessary for the cAMP-dependent inhibition of NHE3. In the present study, we characterized the interaction between NHE3 and E3KARP using in vitro binding assays. We found that NHE3 directly binds to E3KARP and that the entirety of the second PSD-95/Dlg/ZO-1 (PDZ) domain plus the carboxyl-terminal domain of E3KARP are required to bind NHE3. E3KARP binds an internal region within the NHE3 C-terminal cytoplasmic tail, defining a new mode of PDZ domain interaction. Analyses of cellular distribution of NHE3 and E3KARP expressed in PS120 fibroblasts show that NHE3 and E3KARP are co-localized on the plasma membrane, but not in a distinct juxtanuclear compartment in which NHE3 is predominantly expressed. The distributions of NHE3 and E3KARP were not affected by treatment with 8-bromo-cAMP. As shown earlier for the human homolog of NHERF, we also found that the cytoskeletal protein ezrin binds to the carboxyl-terminal domain of E3KARP. These results are consistent with the possibility that E3KARP and NHERF may function as scaffold proteins that bind to both NHE3 and ezrin. Since ezrin is a protein kinase A anchoring protein, we suggest that the scaffolding function of E3KARP binding to both ezrin and NHE3 localizes cAMP-dependent protein kinase in the vicinity of the cytoplasmic domain of NHE3, which is phosphorylated by elevated cAMP.
Highlights
NHE3 phosphorylation by PKA was initially demonstrated by Moe et al [3], who showed that in AP-1 cells, a Chinese hamster ovary cell line, the cAMP-elicited inhibition of NHE3 activity was accompanied by an increase in NHE3 phosphorylation level
We found that the interaction of NHE3 with E3KARP represents a new mode of PDZ binding and that it requires a PDZ domain and a non-PDZ domain of E3KARP
To ensure that the tagging of E3KARP protein with His6 and S-protein did not alter its function in cAMP-dependent inhibition of NHE3, we studied the activity of NHE3V in response to 100 M 8-Br-cAMP
Summary
We previously reconstituted the cAMP-dependent inhibition of NHE3 by transfecting one of two regulatory proteins, E3KARP (NHE3 kinase A regulatory protein; named TKA-1) or NHERF (NHE regulatory factor), in the PS120 fibroblast cell line, which lacks these regulatory proteins, and cAMP does not affect NHE3 activity [5,6,7]. We found that E3KARP binds to an internal domain within the NHE3 cytoplasmic tail, which has previously been shown to be essential for PKA-dependent inhibition of NHE3 [13]. We show that E3KARP binds to the cytoskeletal protein ezrin, which was previously shown to bind to PKA These findings suggest a potential model for E3KARP in the inhibition of NHE3 activity by PKA.
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