Betaine plays an important role in protecting cells from hypertonic stress. The renal medulla is normally hypertonic and the degree of hypertonicity changes depending on hydration status. In some pathologic states all tissues experience hypertonicity and adapt to the adverse environment using mechanisms similar to those of the renal medulla. Regulation of the betaine transporter is critical in osmo-protective accumulation of betaine in renal epithelial cells. When cells are exposed to a hypertonic environment, transcription of the gene coding for the betaine transporter is markedly stimulated resulting in increased activity of the betaine transporter and betaine accumulation. The betaine transporter is also regulated post-translationally by activators of protein kinases A and C. In order to understand how cells sense hypertonic stress and how the signal is transduced to the transcriptional machinery, the transcription factor that mediates transcriptional stimulation of the betaine transporter gene in response to hypertonicity will be cloned. A genetic selection strategy using yeast, an affinity screening, or amino acid sequence obtained from the purified protein will be used to clone the cDNA for the transcription factor. Antibodies to the transcription factor will be raised based on the cDNA sequence. The cDNA and antibody probes will be used to study how the transcription factor is activated by hypertonicity. If the regulation is at the level of transcription as determined by Northern blot and nuclear run-on assays, the mechanism of transcriptional regulation will be studied by cloning the gene for the transcription factor and searching for regulatory sequence elements. Regulation at the level of protein abundance, shift in subcellular localization, and phosphorylation will be also studied using the antibodies. To learn how protein kinases inhibit activity of the betaine transporter, the effects of activation of the protein kinases on changes in phosphorylation and subcellular localization of the betaine transporter will be studied using specific antibodies.
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