The steroid hormone control of posttranslational processes, such as protein trafficking, is a poorly understood but potentially important mechanism to facilitate coordinated and intricate changes in plasma membrane composition, function, and cell-cell interactions. Glucocorticoids, one class of steroid hormones, stimulate tight junction formation and cell polarity in cultured monolayers of nontransformed and transformed mammary epithelial cells, implicating this lactogenic steroid as a key in vivo regulator of cell-cell contact during differentiation of the mammary gland. Glucocorticoids regulate the intracellular recruitment of the endogenous, or transfected exogenous, tight junction- associated ZO-1 phosphoprotein from a cytoplasmic compartment to the plasma membrane, and, co-immunoprecipitations have detected several glucocorticoid-regulated ZO-1 binding proteins. To elucidate the molecular basis of this unique steroid-regulated response, in vitro mutagenesis of ZO-1, in combination with confocal microscopy of transfected cells, will be used to map the precise structural domains within ZO-1 required for its glucocorticoid-regulated targeting to the plasma membrane. As a complementary biochemical approach, purified fusion proteins containing specific ZO-1 coding domains will be introduced into permeabilized mammary tumor cells and tested for their glucocorticoid regulated trafficking to the plasma membrane and/or their ability to disrupt the localization of endogenous ZO-1. Finally, interactive cloning strategies will be employed to isolate and identify the glucocorticoid regulated ZO-1-binding proteins uncovered by coimmunoprecipitation, determine their ZO-1 domain binding specificity and functionally characterize their role in the glucocorticoid regulated signaling pathway controlling tight junction dynamics. Cell-cell interactions play a critical role in controlling the growth and differentiation of normal tissue, whereas, the malignant phenotype is associated with disruptions in this process. The overall goal is to understand the mechanism by which glucocorticoid-regulated posttranslational pathways control cell-cell interactions in normal and transformed mammary cells. This information may eventually prove to be clinically useful in the potential development of therapeutic strategies to control physiologic disorders and neoplasia with a apparent alterations in protein trafficking , plasma membrane composition and/or cell-cell interactions.
