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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022742-05
Application #
3134279
Study Section
Immunobiology Study Section (IMB)
Project Start
1985-08-01
Project End
1990-07-31
Budget Start
1989-08-01
Budget End
1990-07-31
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Kansas
Department
Type
Schools of Medicine
DUNS #
016060860
City
Kansas City
State
KS
Country
United States
Zip Code
66160
Chen, T Y; Lei, M G; Suzuki, T et al. (1992) Lipopolysaccharide receptors and signal transduction pathways in mononuclear phagocytes. Curr Top Microbiol Immunol 181:169-88
Novotney, M; Chang, Z L; Uchiyama, H et al. (1991) Protein kinase C in tumoricidal activation of mouse macrophage cell lines. Biochemistry 30:5597-604
Suzuki, T (1991) Signal transduction mechanisms through Fc gamma receptors on the mouse macrophage surface. FASEB J 5:187-93
Yamada, A; Suzuki, T (1989) Fc gamma 2b receptor-mediated phagocytosis by a murine macrophage-like cell line (P388D1) and peritoneal resident macrophages. Up-regulation by the inhibitors of phospholipase A2 and cyclooxygenase. J Immunol 142:2457-63
Adachi, I; Connolly, N; Suzuki, T (1989) Interferon-gamma down-regulates membrane adenylate cyclase activity of a murine macrophage-like cell line (P388D1). Cytokine 1:36-44
Kagami, M; Funatsu, Y; Suzuki, T (1989) Production and characterization of monoclonal antibodies to Fc gamma 2a-binding protein isolated from the detergent lysate of a murine macrophagelike cell line, P388D1. J Leukoc Biol 45:311-21
Yamada, A; Dileepan, K N; Stechschulte, D J et al. (1989) Regulation of Fc gamma 2a receptor-mediated phagocytosis by a murine macrophage-like cell line, P388D1: involvement of casein kinase II activity associated with Fc gamma 2a receptor. J Mol Cell Immunol 4:191-9;discussion 199-201
Shamma, M S; Fernandez-Botran, R; Suzuki, T (1988) PGE2-induced desensitization of adenylate cyclase of a murine macrophage-like cell line (P388D1). Prostaglandins 36:329-41
Yamamoto, H; Suzuki, T (1987) Prostaglandin-E2-induced activation of adenosine 3'-5' cyclic monophosphate-dependent protein kinases of a murine macrophage-like cell line (P388D1). J Immunol 139:3416-21