NHE3 is the Na + /H + exchanger located on the intestinal and renal brush border membrane, where it functions in transepithelial Na + absorption. The brush border Na + absorptive process is acutely inhibited by activation of cAMP-dependent protein kinase, but the molecular mechanism of this inhibitory effect is poorly understood. We have identified two regulatory proteins, E3KARP and NHERF, that interact with NHE3 to enable cAMP to inhibit NHE3. The two regulatory proteins are structurally related, sharing ≈50% identity in amino acid sequences. It has been previously shown that when NHE3 is transfected into PS120 fibroblasts or Caco-2 cells, cAMP failed to inhibit NHE3 activity. Northern blot analysis showed that both PS120 and Caco-2 cells lacked the expression of both E3KARP and NHERF. In contrast, other cell lines in which cAMP inhibits NHE3, including OK, CHO, and LLC-PK 1 cells, expressed NHERF-related regulatory proteins. To determine their functions in cAMP-dependent inhibition of NHE3, E3KARP and NHERF were transfected into PS120/NHE3 fibroblasts. Transfection in PS120/NHE3 fibroblasts with either NHERF or E3KARP reconstituted cAMP-induced inhibition of NHE3, resulting in 25–30% inhibition in these cells.

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 carboxylterminal 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.NHE3 is the Na ϩ /H ϩ exchanger in the brush border membrane of the proximal tubule and small intestine and colon that plays a major role in transepithelial Na ϩ absorption (1, 2). Among the various stimuli that modulate NHE3 activity (1), cAMP inhibits the transepithelial Na ϩ absorption by NHE3. However, the molecular mechanisms of the cAMP-dependent inhibition of the Na ϩ absorptive process remain unknown. Currently, two major mechanisms have been suggested for the cAMP-dependent inhibition of NHE3 activity. The first involves an increase in NHE3 phosphorylation level by PKA, 1 and the second requires the presence of a regulatory factor. 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. More recently, Kurashima et al. (4) identified the sites of phosphorylation in NHE3 by PKA in AP-1 cells. In contrast, we previously reconstituted the cAMP-dependent inhibition of NHE3 by transfecting one of two regulatory proteins, E3KARP (NHE3 kinase A regulatory protein 2 ; also named TKA-1) or NHERF (NHE regulatory factor), in the PS120 fibroblast cell line, which lacks these regulatory proteins, and consequently, cAMP does not affect NHE3 activity (5-7). This demonstrates the necessity of a regulatory protein in cAMP-dependent inhibition of NHE3. E3KARP was cloned from a human lung library by yeast two-hybrid screening ...

NHE3 is the apically located Na؉ /H ؉ exchanger in the gut and in the renal proximal tubule. Acute inhibition of this transporter by cAMP requires the presence of either of two NHE3-associated proteins, NHERF or E3KARP. It has been suggested that these proteins either directly regulate NHE3 activity after being phosphorylated by protein kinase A (PKA) or that they may serve as adapters that localize PKA near NHE3. We studied the role of NHERF and E3KARP in opossum kidney cells, which endogenously express NHE3, NHERF, and ezrin and display cAMP-dependent inhibition of NHE3. In vivo phosphorylation studies showed that NHERF is a phosphoprotein under basal conditions, but does not change its phosphorylation state after 8-bromo-cAMP treatment, and that E3KARP is not phosphorylated at all. Co-immunoprecipitation showed that NHERF and E3KARP bind both NHE3 and ezrin. Using cAMP analogs it was demonstrated that NHE3 activity, measured as sodium-dependent recovery of the intracellular pH after intracellular acidification, is inhibited by PKA type II. Because others have shown that ezrin binds PKA type II and that NHE3 is phosphorylated by PKA we suggest that NHERF and E3KARP are adapters that link NHE3 to ezrin, thereby localizing PKA near NHE3 to allow NHE3 phosphorylation. NHE31 is the apically located Na ϩ /H ϩ exchanger isoform that together with a Cl Ϫ /HCO 3 Ϫ exchanger or a Na ϩ /HCO 3 Ϫ co-transporter, respectively, mediates the majority of NaCl or NaHCO 3 absorption in the ileum and proximal colon and in the proximal tubule (1, 2). Cyclic AMP is one of the major intracellular messengers mediating the inhibition of NHE3 (1, 3). Two models of how NHE3 is inhibited by cAMP have evolved: the first model proposes that NHE3 is regulated through direct phosphorylation of the transport protein. This model is based on findings that in AP1 cells treatment with 8-Br-cAMP results in the phosphorylation of NHE3 that parallels the inhibition of transport activity (4, 5). The other model proposes that regulatory proteins are required to transduce cellular signals between protein kinase A and NHE3 (6, 7). The existence of one or more regulatory proteins was suggested based on the finding that in solubilized rabbit renal brush-border a protein fraction that was required for regulation of NHE3 could be separated from NHE3 itself (6), and that in PS120 fibroblasts several signals that regulate NHE3 did not change the phosphorylation state of NHE3 (7). It was speculated that such regulatory proteins would be the substrate for protein kinases (6, 7) and that they would interact with NHE3 in a phosphorylation dependent manner, resulting in a change of NHE3 activity. It is not known whether these two mechanisms are independent processes or complementary in that both the regulatory proteins and NHE3 are phosphorylated in response to cAMP. Two closely related regulatory proteins of NHE3, NHERF (NHE regulatory factor), and E3KARP (NHE3 kinase A regulatory protein), have recently been identified (8, 9). We previously showed that there is...