MOLECULAR MECHANISM OF REGULATION OF NA/H EXCHANGER NHE3

University Of Texas Hlth Sci Ctr Houston, Houston TX

Investigators

Abstract

DESCRIPTION (adapted from the application) (This Description is submitted for R03 Award Application as a supplement for K08 Award DK02557).This proposal deals with short-term regulation by protein kinase C (PKC) and growth factors of the brush border (BB) Na/H exchanger NHE3 in the human intestinal 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 growth factors (acting through a tyrosine kinase receptor) and inhibited by phorbol ester (PMA; via activation of PKC). Most of this 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 acute regulation of the exchanger activity by growth factors and PKC in the intestinal epithelium, and that this process is mediated by the NHE3 C-terminus. The PS120 fibroblasts and, ultimately, polarized human carcinoma cell line Caco-2 transfected with native or mutated NHE3 cDNA will be used as experimental model. In the initial stage of the original proposal, we examined the cellular redistribution of NHE3 in response to stimulation of PKC using digital morphometric analysis at the level of confocal fluorescent microscopy, and reversible surface biotinylation. Next, we engineered and stably transfected into PS120 fibroblasts a fusion of NHE3 and green fluorescent protein (NIHE3-GFP) cDNA, which enabled us to simultaneously quantitate changes in the activity (fluorescent method with SNARF-1) and kinetics of intracellular trafficking of NHE3-GFP (digital confocal morphometric analysis) in response to bFGF at the level of a single living cell. We now propose to extend these studies into stably transfected Caco-2 cells. To define the role of C-terminus in regulation of the intracellular trafficking of NHE3, we will examine the effect of specific mutations of NHE3 C-terminal sequences suspected of being involved in PMA-mediated removal of the exchanger from the plasma membrane (LL, PXXP and YXX-phi motifs). Finally, we will investigate the role of a critical 104 aa sequence of the NHE3 C-terminus which is required for PMA-mediated inhibition of the exchanger by constructing a chimera in which this domain will be replaced by a corresponding domain from another NHE isoform, NHE2, which is stimulated by PMA. The insights in the mechanism of regulation of NEH3 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.