PHYSIOLOGY OF PITUITARY CELL GLUCOCORTICOID BINDING

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The ultimate objective of this project is to understand the role of glucocorticoid binding and the physiology of hormone action. Toward this end, we have studied several aspects of glucocorticoid receptor physiology, including the kinetics of receptor bind, activation, and nuclear translocation. Progress has been made on three objectives of this project: isolation of ACTH mRNA and production of ACTH cDNA, analysis of in vivo receptor activation and nuclear binding of the glucocorticoid receptor, and characterization of the glucocorticoid receptor itself. We have characterized approximately 1200 nucleotide cDNA to ACTH mRNA cloned into p8R322. This clone has been sequenced, used to identify ACTH mRNA and polysomal RNA extracted from AtT-20 cells, and used by another laboratory at Vanderbilt to identify and characterize ACTH mRNA from ectopic hormone-producing tumors of humans. The extent of in vivo activation and nuclear translocation produced in AtT-20 cells by four glucocorticoid agonists has been determined. This marks the first time that receptor activation and nuclear translocation have been correlated in vivo. It was found for dexamethasone, triamcinolone acetonide, prednisolone, and corticosterone that there was a rigid correlation between binding affinity, activation, and nuclear bind. Furthermore, although the extent of activation varied with each steroid, the fraction of activated receptor that was translocated was similar (approximately 60%) in each case, lending support to the concept that nuclear translocation is a simple partitioning phenomenon. Last, the rat glucocorticoid receptor has been purified and monoclonal antibodies have been produced to it that also recognize the mouse glucocorticoid receptor. These reagents will make further analysis of the mouse glucocorticoid receptor possible. As of this writing, stable hybridomas producing monoclonal antibodies to the glucocorticoid receptor have not been described. During the past year progress has been made in several areas including: (1)\immunopurification of the mouse and rat glucocorticoid receptors and their proteolytic fragments; (2)\isolation of the mouse glucocorticoid receptor gene; and (3)\identification of the subunit composition of the native glucocorticoid receptor. Specific objectives for the coming year are: (1) to isolate the mouse glucocorticoid receptor gene, using a monoclonal antibody to the receptor developed in this laboratory; (2) to characterize the relationship between intact cell uptake and biological potency of steroids of various potency; and (3)\to purify the mouse and rat glucocorticoid receptor with immunoaffinity techniques. (D)