Experiments have been designed to study the interactions between cellular components and a unique, intracellular class of lymphocyte stimulator, the 7,8-disubstituted guanine ribonucleosides. While encouraging results with these compounds in studies of cells from patients with Common Variable Immunodeficiency, IgA deficiency, AIDS-related complex and AIDS suggest that they may have significant clinical potential, little is known about their mechanism of action. The first major objective proposed is to evaluate the biological relevance of the binding proteins as receptors. The relevance of proteins radiolabeled by biologically active photoaffinity ligands will be determined in competition studies, and by examining hyporesponsive, nonresponsive, and inducible cell types. Monoclonal antibodies to these proteins will be raised and their specificity verified by Western blot. These antibodies will be micro-injected into cells to antagonize or trigger biological responses. Purified receptor proteins will be used to reconstitute nucleoside responsiveness in hyporesponsive, nonresponsive, and inducible cells. Affinity-purified proteins from normal mice will be examined for binding affinity and binding site topology, and compared with proteins from hyporesponsive and nonresponsive strains. The second major objective is to determine the physico-chemical properties of purified (receptor) proteins, including Mr, isoelectric point, degree of glycosylation and linkages involved. The relationship between the two forms of binding protein will be explored in cells from normal and hyporesponsive strains. Proteolytic cleavage fragments will be separated and microsequenced to elucidate the overall protein sequences. The third major objective is to study interactions between the nucleoside and its receptor. The functional topology of ligand recognition by the receptor will be studied for high and low affinity receptors with an array of biologically inactive analogs. Similarly, binding domains on the receptor proteins will be defined. Finally, nucleoside receptors will be investigated within the context of the cell. Immuno-localization of receptors in resting and activated cells, translocation to different cellular compartments, cycling, and degradation will be studied with the electron microscope. Regulation of receptor synthesis and turnover will be compared to functional status. Effects of B cell agonists and lymphokines on receptor expression will be examined throughout the cell cycle in responder, nonresponder and inducible cell types.
