PROSTAGLANDIN SENSITIVE ADENYLATE CYCLASE

我们研究的长期目标是阐明生物化学 最初由特定的信号触发的一系列信号的性质 配体与表面受体的结合最终导致 调节参与免疫反应的细胞的功能。我们会 在这项建议中，我们将重点放在生化和生物方面 小鼠PG受体和腺苷环化酶系统的研究 巨噬细胞样细胞系P338D1。 我们的初步研究结果表明，一只小鼠 巨噬细胞样细胞系P388D1具有典型的腺苷酸环化酶 可被PGs、异丙肾上腺素、NaF、GTP或ITS刺激的系统 非水解性类似物GPP(NH)p及不同烷基化反应的抑制作用 试剂。随后，我们证明了完整的细胞既具有高密度 以及它们表面上的低亲和力PGE2结合部位，以及PGE2结合 蛋白质，其大小均匀，电荷适当，并表现出 未被分离程序改变的特定PGE2结合属性可以是 通过亲和层析从细胞的洗涤剂裂解物中获得 PGE2共价偶联到AH-琼脂糖凝胶上。拟议的研究是一项 这些初步观察的延伸，以进一步表征 P388D_1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机制 系统。具体地说，我们将尝试：1)将光亲和标记PG 用于验证分离的PGE2结合蛋白代表PG的受体 受体；2)明确PGE2受体是否定位 主要在细胞表面；3)描绘分子性质 PGE2受体；4)产生针对PGE2的单抗 5)探讨前列腺素E_2诱导的脱敏机制； 6)研究γ-干扰素调控的生化机制。 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.