The steroid hormone control of posttranslational protein sorting is a potentially important means for hormonal regulation of cellular function. Glucocorticoid hormones regulate the localization of cell surface mouse mammary tumor virus (MMTV) glycoproteins in viral-infected rat hepatoma cells by inducing components within a previously unknown and genetically distinct posttranslational protein trafficking pathway. This observation implies that physiological regulators, such as steroid hormones, can affect the expression or activity of functioning protein products long after they have been transcribed. Cell fusion and DNA rescue studies using a unique sorting variant defective in the steroid-regulated sorting response suggest the existence of a new class of glucocorticoid-regulated trafficking activities (denoted GRT) that are encoded by both rat and human hepatoma cells. 1) The GRT gene will be functionally cloned from a human placental cosmid library by DNA rescue of the sorting variant. Restoration of the inducible trafficking response will be monitored by a transient immunofluorescence assay for cell surface MMTV glycoproteins. 2) The GRT genomic sequences will be used as a probe to isolate full length GRT cDNA from a hepatoma cDNA library. The GRT cDNA will be sequenced to establish identity and employed as a hybridization probe to examine the glucocorticoid-regulated expression of the GRT gene. 3) To define potential sorting signals for the glucocorticoid-regulated trafficking pathway, precise segments of cloned MMTV glycoprotein genes will be mutated in vitro and the glucocorticoid-regulated sorting of altered glycoproteins examined in transfected hepatoma cells. 4) Hybrid glycoprotein genes will be constructed of discrete regions of the MMTV glycoprotein gene and the human insulin receptor and the ability of these MMTV glycoprotein domains to direct entry of the hybrid glycoproteins into the glucocorticoid-regulated sorting pathway will be monitored after DNA transfection into wild type and variant hepatoma cells. 5) Long term objectives are to investigate the cellular substrates and tissue-specific expression of the GRT activity. The information from these studies should significantly contribute to understanding fundamental hormonal regulatory mechanisms acting posttranslationally on protein trafficking as well as elucidating host cell-derived factors that may control retrovirus protein expression and virion assembly.
