We have demonstrated the presence of a binding protein for CBG on the surface of glucocorticoid target cells and on rat liver and kidney membranes. This protein is highly specific for CBG and binds it with an affinity of 1 uM. This is also the approximate level of circulating CBG. Previous studies have demonstrated the presence of intracellular CBG. The presence of CBG within target cells and a mechanism by which CBG can bind to these cells and perhaps be internalized suggest that this plasma steroid binding protein has a more complex physiologic role than has previously been believed. This proposal is designed to characterize this protein and its interaction with CBG. Binding studies will employ both isolated membranes from rat tissues and FAO cells (a rat hepatoma cell line). The pH, steroid, and temperature dependence of binding will be determined. The tissue distribution of this binding site will be determined as will its developmental appearance. The ability of cells to internalize CBG and the pathway by which this is accomplished will be determined by pronase digestion after binding and by electron micrographic autoradiographic studies at various time points. The effect of various inhibitors upon internalization will also be determined. We will perform studies to solubilize the binding protein and will attempt to achieve its purification. The effect of CBG binding upon two second messenger systems (cAMP generation and phosolipid turnover) will be determined. CBG could serve to fine tune the ability of the cell to respond to subsequent hormone administration. We will examine this by adding CBG to culture of FAO cells and determining whether this has an effect on either the dose response or maximal degree of induction of tyrosine amino transferase by dexamethasone. Whether CBG serves only to transport steroid or has other roles, the control of its circulating level is physiologically important. The site(s) of synthesis of CBG will be determined by examining whether tissue slices or isolated messenger RNA from those tissues can direct its synthesis. Hormonal manipulation of animals and/or organ cultures will be used to determine the factors controlling its synthesis. These studies will help to determine the physiologic role of CBG and its specific membrane binding protein.
