Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 approximately 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-PK1 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.
 ...
PMID:cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein. 909 37

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.
 ...
PMID:NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin. 974 60

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase- and ATP-regulated chloride channel, the activity of which determines the rate of electrolyte and fluid transport in a variety of epithelial tissues. Here we describe a mechanism that regulates CFTR channel activity, which is mediated by PDZ domains, a family of conserved protein-interaction modules. The Na(+)/H(+) exchanger regulatory factor (NHERF) binds to the cytoplasmic tail of CFTR through either of its two PDZ (PDZ1 and PDZ2) domains. A recombinant fragment of NHERF (PDZ1-2) containing the two PDZ domains increases the open probability (P(o)) of single CFTR channels in excised membrane patches from a lung submucosal gland cell line. Both PDZ domains are required for this functional effect, because peptides containing mutations in either domain are unable to increase channel P(o). The concentration dependence of the regulation by the bivalent PDZ1-2 domain is biphasic, i.e., activating at lower concentrations and inhibiting at higher concentrations. Furthermore, either PDZ domain alone or together is without effect on P(o), but either domain can competitively inhibit the PDZ1-2-mediated stimulation of CFTR. Our results support a molecular model in which bivalent NHERF PDZ domains regulate channel gating by crosslinking the C-terminal tails in a single dimeric CFTR channel, and the magnitude of this regulation is coupled to the stoichiometry of these interactions.
 ...
PMID:Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction. 1115 44

Sodium-hydrogen exchanger regulatory factor isoform-1 (NHERF-1) and NHERF-2 are two structurally related PDZ-domain-containing protein adapters that effectively transduce cyclic AMP (cAMP) signals that inhibit NHE3, the sodium-hydrogen exchanger isoform present at the apical surface of kidney and gut epithelia. The mouse renal proximal tubule expresses both NHERF isoforms, suggesting their redundant functions as regulators of renal electrolyte metabolism. To define the role of NHERF-1 in the physiological control of NHE3, we analyzed NHE3 activity in isolated brush border membrane (BBM) preparations from renal proximal tubules of wild-type (WT) and NHERF-1 (-/-) mice. Basal Na(+)-H(+) exchange was indistinguishable in BBMs from WT and NHERF-1 (-/-) mice (0.96+/-0.08 and 0.95+/-0.10 nmol/mg protein/10 s, respectively). Activation of membrane bound cAMP-dependent protein kinase (PKA) by cAMP inhibited NHE3 activity in WT BBMs (0.55+/-0.07 nmol/mg protein/10 s or 40+/-9%, P<0.01) but had no discernible effect on Na(+)-H(+) exchange in the NHERF-1 (-/-) BBM (0.97+/-0.07 nmol/mg protein/10 s; P=not significant). This was associated with a significant decrease in cAMP-stimulated phosphorylation of NHE3 immunoprecipitated from solubilized NHERF-1 (-/-) BBMs. As the protein levels for NHE3, NHERF-2, PKA and ezrin were not changed in the NHERF-1 (-/-) BBMs, the data suggest a unique role for NHERF-1 in cAMP-mediated inhibition of NHE3 activity in the renal proximal tubule of the mouse.
 ...
PMID:NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse renal apical membranes. 1258 53

Disorganized ion transport caused by hypo- or hyperfunctioning of the cystic fibrosis transmembrane conductance regulator (CFTR) can be detrimental and may result in life-threatening diseases such as cystic fibrosis or secretory diarrhea. Thus, CFTR is controlled by elaborate positive and negative regulations for an efficient homeostasis. It has been shown that expression and activity of CFTR can be regulated either positively or negatively by PDZ (PSD-95/discs large/ZO-1) domain-based adaptors. Although a positive regulation by PDZ domain-based adaptors such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple adaptor interactions, are not known. Here we demonstrate a physical and physiological competition between EBP50-CFTR and Shank2-CFTR associations and the dynamic regulation of CFTR activity by these positive and negative interactions using the surface plasmon resonance assays and consecutive patch clamp experiments. Furthermore whereas EBP50 recruits a cAMP-dependent protein kinase (PKA) complex to CFTR, Shank2 was found to be physically and functionally associated with the cyclic nucleotide phosphodiesterase PDE4D that precludes cAMP/PKA signals in epithelial cells and mouse brains. These findings strongly suggest that balanced interactions between the membrane transporter and multiple PDZ-based adaptors play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues.
 ...
PMID:Dynamic regulation of cystic fibrosis transmembrane conductance regulator by competitive interactions of molecular adaptors. 1724 9