cAMP-mediated inhibition of the epithelial brush border Na ⁺ /H ⁺ exchanger, NHE3, requires an associated regulatory protein

Abstract

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.

Lysophosphatidic acid prevents apoptosis of Caco-2 colon cancer cells via activation of mitogen-activated protein kinase and phosphorylation of Bad

“…LPA 2 has carboxyl-terminal motifs that allow interaction with a PDZ (PSD-95/DlgA/ZO-1) domain of the Na + /H + exchanger regulatory factor 2 (NHERF2), which functions as a scaffold to link signaling proteins, such as PKA and PKCα, to GPCRs and transporters at the cell surface [21,23,[29][30][31]. NHERF2 links LPA 2 to PLC-β3 and hence affects the activation of downstream targets, such as Erk and cyclooxygenase-2 [23].…”

Altered cGMP Dynamics at the Plasma Membrane Contribute to Diarrhea in Ulcerative Colitis

“…cAMPdriven inhibition of NHE3 can be mediated via NHERF1, NHERF2, and NHERF3 (PDZK1), whereas cGMP-mediated inhibition was found to specifically require NHERF2. 28,57 NO was found to inhibit NHE3 function by the NO-cGMP pathway, but without account for the effect of inducible NO under inflammation conditions for NHE3 regulation. 40,58 In another study, NHE3 expression was found to be unaltered in cytokine-induced inflammation in interleukin-10edeficient mice marked by up-regulated iNOS levels.…”

“…54e56 It is interesting that the same intracellular cAMP, Ca 2þ , or cGMP that activates CFTR would inhibit NHE3. 55,57 Not only do CFTR and NHE3 share the same secondary messengers for regulation but also regulatory scaffolding NHERF proteins. 48 NHE3 harbors a C-terminal PDZ-binding motif, similar to CFTR that would mediate NHE3 interaction with other membrane proteins, cytoskeleton, and protein kinases through NHERFs.…”

Postnatal developmental expression of the PDZ scaffolds Na+-H+ exchanger regulatory factors 1 and 2 in the rat cochlea

“…NHERFs also serve to recruit signaling molecules, such as PKA, PKC, and PI-3 kinase, to the receptor complexes (Fanning and Anderson 1999;Shenolikar and Weinman 2001;Yun 2003;Shenolikar et al 2004;Lee-Kwon et al 2005;Weinman et al 2005). Importantly, although NHERF-1 and NHERF-2 were originally described in the kidney, Northern blot analysis shows an overlapping and wide-spread distribution pattern for NHERF-1 and NHERF-2 mRNAs in human tissues, including brain, heart, colon, small intestine, liver, prostate, spleen, and placenta (Weinman et al 1995;Yun et al 1997). Immunohistochemical studies have demonstrated NHERF-1 and NHERF-2 in the human (Weinman et al 2002) and mouse (Wade et al 2003) kidney, NHERF-1 in the mouse retina (Nawrot et al 2004) and Schwann cell processes (Gatto et al 2003), and NHERF-2 in endothelia and pericytes from rat descending vasa recta (Lee-Kwon et al 2005).…”

“…NHERF-1 (also called EBP50) was the first scaffold identified for an epithelial transporter and was found to mediate protein kinase A (PKA) inhibition of NHE3 in renal brush border vesicles (Weinman et al 1995). Subsequently, its close relative NHERF-2 (also called E3KARP) was cloned and characterized (Yun et al 1997). NHERF-1 and NHERF-2 consist of 358 and 337 amino acids, respectively, sharing 54% homology and possess two PDZ domains and a C-terminal ezrin-radixin-moesinmerlin (ERM)-binding domain that can interact with the actin cytoskeleton (Fanning and Anderson 1999;Shenolikar and Weinman 2001;Wade et al 2003;Yun 2003;Shenolikar et al 2004).…”