(taken from application) This proposal deals with short term regulation by protein kinase C and growth factors of the brush border (BB) Na+/H+ exchanger NHE3 in the human gut epithelium. NHE3 is involved in neutral NaCl absorption between meals in the mammalian small intestine, and its inhibition plays an important role in pathogenesis of diarrheal diseases. Its activity is stimulated by EGF (acting by a tyrosine kinase receptor) and inhibited by phorbol ester (PMA; via activation of PKC). All the regulation occurs within minutes via Vmax mechanism which suggests the effect on either the turnover number, or the number of active molecules at the BB (recruitment vs. removal). The hypothesis of this proposal is that BB recruitment / removal of NHE3 plays an important role in the mechanism of the regulation of the exchanger activity by growth factors and PKC in the gut epithelium, and that this process is mediated by the NHE3 C-terminus. The polarized human carcinoma cell line Caco 2 transfected with either full length (NHE3 V) or mutated NHE3 cDNA will be used as experimental model. First, quantitative evaluation the BB recruitment and removal of NHE3 V in response to EGF and PMA will be performed using computerized morphometry at the level of confocal fluorescent microscopy and reversible surface biotinylation. We will then engineer and transfect into Caco 2 cells a fusion of NHE3 and green fluorescent protein (NHE3-GFP) cDNA, which would enable us to simultaneously quantitate the activity (microscopic fluorescence method with SNARF-1) and intracellular trafficking of NHE3-GFP (confocal morphometric analysis) in response to EGF and PMA at the level of single living cell. To define the role of the C-terminus in this regulation, the effect of specific mutations of NHE3 C-terminal sequences suspected of being involved in PMA-mediated BB removal of the exchanger will be examined. Finally, we will investigate the role of a critical 104 aa sequence of the NHE3 C-terminus required for PMA-mediated inhibition by constructing a chimera in which this domain is replaced by a corresponding domain from another NHE isoform, NHE2, which is stimulated by PMA. The insights in the mechanism of regulation of NHE3 to be gained from these studies will substantially aid our understanding of digestive physiology, and pathobiology of diarrheal diseases in which changes in regulation of NaCl absorption occur.
| Singh, Sharda P; Janecki, Andrzej J; Srivastava, Sanjay K et al. (2002) Membrane association of glutathione S-transferase mGSTA4-4, an enzyme that metabolizes lipid peroxidation products. J Biol Chem 277:4232-9 |
| Janecki, A J; Janecki, M; Akhter, S et al. (2000) Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts. J Histochem Cytochem 48:1479-92 |
| Janecki, A J; Janecki, M; Akhter, S et al. (2000) Basic fibroblast growth factor stimulates surface expression and activity of Na(+)/H(+) exchanger NHE3 via mechanism involving phosphatidylinositol 3-kinase. J Biol Chem 275:8133-42 |
