The long term objective of our studies is to elucidate the biochemical nature of a series of signals which is initially triggered by specific binding of ligand to surface receptor and leads ultimately to the regulation of functions of cells involved in the immune responses. We will focus our efforts in this proposal on the biochemical and biological studies of PG receptors and adenylate cyclase system of a murine macrophage-like cell line, P338D1. Results of our preliminary studies demonstrated that a murine macrophage-like cell line, P388D1, possesses a typical adenylate cyclase system which can be stimulated by PGs, isoproterenol, NaF, GTP or its nonhydrolyzable analog-Gpp(NH)p and inhibited by various alkylating reagents. Subsequently, we have shown that intact cells possess both high and low affinity PGE2-binding sites on their surface, and that PGE2-binding protein, which is homogeneous in size and charge proper ties, and exhibits specific PGE2-binding properties unaltered by isolation procedure, can be obtained from the detergent lysate of the cells by affinity chromatography with PGE2 covalently coupled to AH-Sepharose. The proposed research is an extension of these preliminary observations to further characterize biochemical and biological properties of PGE2 receptors of P388D1 cells and the mechanisms of PG receptor-mediated activation of the adenylate cyclase system. Specifically, we will attempt: 1) to photoaffinity label PG receptors to verify that the isolated PGE2-binding protein represent PG receptors; 2) to clarify whether or not PGE2 receptors are localized primarily on the cell surface; 3) to delineate the molecular properties of PGE2 receptor; 4) to produce monoclonal antibodies specific for PGE2 receptor; 5) to investigate the mechanisms of PGE2-induced desensitization; 6) to examine biochemical mechanisms of modulation of the IFNGamma-induced Ia antigen expression.
