The proposed work wil focus on two proteins, the glucocorticoid receptor and ligandin. Since the first monoclonal antibodies to these proteins have now been obtained in this laboratory, we propose to develop a repetoire of antibodies in order to quantify the topology of the glucocorticoid receptor and of ligandin. For the receptor, antibodies will be directed, by selective screening procedures, towards the active sites: the steroid binding site and the DNA-binding site as well as to non-active site antigens. The latter will be particularly useful for one-step column chromatographic purification of the receptor. This will permit a host of studies, previously not possible, such as evaluation of direct phosphorylation and dephosphorylation of the receptor as well as other aspects of the activation mechanism. For ligandin, monoclonal antibodies will be developed against the glutathione S-transferase catalytic center, against the non-specific ligand binding site and also against non-active site antigenic groupings. These antibodies will make it possible to localize the genes for ligandin by somatic cell genetic techniques previously not possible with polyclonal antibodies because of unexplained CRMs appearing in a variety of segregating hybrids. Recent experiments have shown a possible relationship between DNA polymerase Alpha and the glucocorticoid receptor. This potential relationship will be explored in detail and studies with other steroid receptors will be included also to determine specificity at the level of the receptor. This work will contribute to our understanding of the mechanism of action of glucocorticoid hormones and of the molecular biology of the ligandin molecule. Monoclonal antibodies to the receptor will make feasible more quantitative techniques for diagnosis of human leukemias which can be treated effectively with glucocorticoids. Monoclonal antibodies to ligandin may provide a new method for detection of specific diseases of human kidney proximal tubular cells.
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