The steroid hormone control of posttranslational protein trafficking is a poorly understood, but potentially important mechanism for hormonal regulation of cellular function. The investigator has uncovered a previously unknown and genetically distinct glucocorticoid-regulated posttranslational response that controls the stable localization of cell surface mouse mammary tumor virus (MMTV) glycoproteins in viral-infected rat hepatoma cells. Glucocorticoids alter the final concentration of cell surface-associated MMTV glycoproteins by decreasing the turnover rate during transport. This novel effect of glucocorticoids on glycoprotein stability suggests the existence of a new class of glucocorticoid-regulated components of a degradative system in the exocytic pathway. A unique uncleavable MMTV glycoprotein target for the steroid regulated degradative system was designed by site-directed mutagenesis of the endoproteolytic site. The investigator lists four Specific Aims of this proposal. 1) Precise segments of the uncleavable MMTV glycoprotein gene will be mutated or substituted in vitro and the glucocorticoid-regulated turnover and cell surface accumulation of the altered glycoproteins examined in transfected hepatoma cells in order to define potential degradative signals for the glucocorticoid-regulated degradative system. 2). Hybrid glycoprotein genes will be constructed of discrete regions of the MMTV glycoprotein gene and the human insulin receptor gene to test the ability of specific MMTV glycoprotein domains to mediate the glucocorticoid-regulated stability response. 3) A permeabilized hepatoma cell system, which reconstitutes MMTV glycoprotein trafficking in vitro and allows the entry of membrane impermeable reagents into the cell, will be used to identify the cellular location and metabolic requirements of the glucocorticoid-regulated degradative system.4) The in vitro reconstitution of MMTV glycoprotein trafficking will be used as an assay to isolate, characterize and identify components of the glucocorticoid regulated degradative system. Eventually, antibody and cDNA probes will be generated to isolate degradative factors to explore their glucocorticoid dependent expression and function.
