The Role of the NHERF-1 and NHERF-2 Adapter Proteins in Intestinal Ion Transport Regulation

The chloride channel CFTR (cystic fibrosis transmembrane conductance regulator) and the sodium/proton exchanger NHE3 are key proteins involved in transepithelial ion and water transport in several epithelial tissues, including the intestine. In this thesis we mainly focus on the role of the NHERF (NHE3 Regulatory Factor) proteins as regulators of CFTR and NHE3 activity. In chapter 2 we show that cAMP- and cGMP-dependent activation of CFTR was moderately reduced in the duodenum and jejunum (but not in ileum) of NHERF-1 deficient mice. This reduced activation of CFTR in may be explained by the ~30% reduced local abundance of CFTR protein in the jejunal crypts of the NHERF-1 deficient mice. As shown in chapter 3, the basal NHE3 activity was decreased in the jejunum and colon of NHERF-1 null mice. In addition, NHERF-1 deficient mice displayed a reduced sodium absorption in the jejunum. These observations may be explained by the reduced NHE3 protein level in the brush border membrane fraction of jejunum and colon of these mice. The cAMP- and/or cGMP dependent inhibition of NHE3 was not appreciably affected in NHERF-1 deficient mice. As described in chapter 4, NHERF-1 and/or NHERF-2 gene knockdown in polarized epithelial cells indicated that NHERF-2 is required for cGKII-dependent regulation of NHE3, but not of CFTR. An overview of the proteome of the jejunal brush border of the mouse is presented in chapter 5. Finally, chapter 6 shows that the glial fibrillary acidic protein (GFAP) is a specific binding partner for cGKII. cGKII was able to phosphorylate GFAP in vitro and expression of active cGKII in cultured glioblastoma cells increased GFAP abundance. We propose that cGKII-mediated phosphorylation of GFAP results in a more stable configuration and a prolonged half-life of this protein.

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