Our goal is to better understand the mechanisms of glucocorticoid action in relation to cellular nutrition. We will continue to investigate the interrelationships between the dietary cofactor Vitamin B6 and glucocorticoid receptor mechanisms in human cells. Observations indicate that physiological changes in the intracellular concentrations of pyridoxal phosphate can dramatically decrease the responsiveness of HeLa S3 cells to glucocorticoid administration. The mechanisms for this effect appear to involve direct modification of the receptor protein resulting in an inhibition of DNA binding as well as the down-regulation of glucocorticoid receptor number. We hypothesize that pyridoxal phosphate may be a physiological antagonist of glucocorticoid action. Therefore we wish to explore further the mechanisms of interaction of pyridoxal phosphate with glucocorticoid receptors.
Four specific aims are proposed to evaluate pyridoxal phosphate/glucocorticoid receptor interrelationships: 1) To evaluate, using dense amino acids and monoclonal antibodies to the receptor, the influence of Vitamin B6 status on the metabolism of the glucocorticoid receptor and potential non-binding precursors; 2) To study the regulation of glucocorticoid receptor mRNA and its stability as a function of cellular B6 status; 3) To evaluate the influence of cellular B6 status on glucocorticoid induced transcription, and; 4) To generate peptide maps of the steroid, DNA, and Vitamin B6 binding domains of purified human glucocorticoid receptor. These studies should thoroughly evaluate the inter-relations of Vitamin B6 and glucocorticoid receptor at the cellular and molecular levels and further our understanding of steroid hormone action.
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