PROSTAGLANDIN SENSITIVE ADENYLATE CYCLASE

我们研究的长期目标是阐明 一系列信号的性质，最初由特定的 配体与表面受体结合，最终导致 调节参与免疫应答的细胞的功能。 我们将 在这项建议中，我们将努力集中在生物化学和生物学方面， 小鼠前列腺素受体和腺苷酸环化酶系统的研究 巨噬细胞样细胞系，P338 D1。 我们的初步研究结果表明， 巨噬细胞样细胞系P388 D1具有典型的腺苷酸环化酶 可被PGs、异丙肾上腺素、NaF、GTP或其 非水解类似物Gpp（NH）p和抑制各种烷基化 试剂 随后，我们已经表明，完整的细胞具有高的 和低亲和力的PGE 2结合位点， 蛋白质，其在大小和电荷性质上是均匀的，并且表现出 特异性PGE 2结合特性不被分离过程改变， 通过亲和层析从细胞的去污剂裂解物中获得 与AH-Sepharose共价偶联的PGE 2。 拟议的研究是一项 这些初步观察结果的扩展，以进一步表征 P388 D1细胞PGE 2受体的生化和生物学特性， PG受体介导的腺苷酸环化酶激活的机制 系统 具体而言，我们将尝试：1）光亲和标记PG 受体，以验证分离的PGE 2结合蛋白代表PG 2）明确PGE 2受体是否定位于 主要在细胞表面; 3）描绘的分子特性， PGE 2受体; 4）产生PGE 2特异性单克隆抗体 5）探讨PGE_2诱导脱敏的机制; 6)为了检查IFN γ诱导的细胞凋亡的调节的生化机制， Ia抗原表达。

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.