ROLE OF DEXRAS1 IN PITUITARY FUNCTION

Auburn University At Auburn, Auburn University AL

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Abstract

DESCRIPTION: (Adapted from the applicant's abstract) Dexras1 is a new gene that was identified in AtT-20 cells. The overall goal of this proposal is to gain a better understanding of Dexrasl as a mediator of glucocorticoid action. Recombinant Dexrasl protein will be characterized for its ability to bind GTP and GDP, and its intrinsic GTPase activity will be determined. The recombinant protein will also be used to make monoclonal antibodies, which will be used for protein identification in this and/or future experiments. Since it appears highly expressed in pituitary and may be a mediator of glucocorticoid-negative feedback regulation of ACTH, the cellular source of Dexrasl in the pituitary will be defined using combined in situ hybridization and immunohistochemistry. To directly address whether this protein is involved in negative feedback, AtT-20 cells will be transfected with plasmid vectors capable of regulating the expression of Dexrasl independent of glucocorticoids. Secretion of ACTH from these transfected cells will be measured to assess whether increased expression of Dexrasl correlates with reduced ACTH secretion, supporting the hypothesis that this protein mediates negative feedback. In a final set of experiments, proteins interacting with Dexrasl will be identified using a far-Western screening technique. Identification of proteins that interact with Dexrasl will be extremely useful to better understand how this molecule functions in glucocorticoid targets and may therefore help identify other cell signaling pathways by which this protein acts. While it is known that many glucocorticoid effects within target cells, including pituitary corticotrophs, require steroid-induced gene transcription and translation, the identity of the gene products mediating subsequent actions is poorly understood. It is conceivable that Dexrasl is an important cellular mediator of glucocorticoid action.