CONTROL OF SURFACTANT SECRETION BY GTP BINDING PROTEINS

肺泡2型肺泡细胞合成和分泌肺表面活性物质 在构成模式和监管模式下。我们已经获得了 GTP结合蛋白参与这一途径的证据 两个层次。首先，几种转导G蛋白 它们将表面受体偶联到负责的效应分子上 用于生成第二信使存在于类型2细胞中 细胞膜和细胞质。G的存在，它耦合了B- 腺酰环化酶的肾上腺素能受体，已被暗示 对其他人的早期研究。我们已经获得了直接的证据 除Gs外，还存在两种Gi。此外，我们 已经表明，直接刺激胃肠道蛋白会导致快速 和戏剧性的表面活性物质分泌。初步证据表明， 给出了通过这一途径实现刺激-反应耦合的基础。 第二，我们鉴定并部分提纯了三个低分子 板层中相对分子质量的ras相关GTP结合蛋白 身体。酵母遗传学和哺乳动物体外实验的最新证据 互补性研究表明，这个家庭的成员 在分泌囊泡分选和胞吐中的关键作用。这个 该项目的目标是识别和描述 GTP结合蛋白参与的生理和分子水平 在2型细胞的刺激-分泌耦合中。这些目标将 通过三个具体目标来追求。1)我们将确定和 2型中存在的转导G蛋白种类的定量 细胞，确定胃肠道蛋白在介导表面活性物质中的作用 分泌和鉴定涉及的第二信使系统， 并检测受体介导的这一通路的激活。2)我们 将从薄层中提纯低分子量GTP结合蛋白 身体，获得部分蛋白质序列，克隆cDNA，产生抗 多肽抗血清，并鉴定其亚细胞分布和 这些蛋白质的生理作用。我们还将确定是否 其他低分子量GTP结合蛋白存在于非 2型细胞的板层体室。3)功能 GTP结合蛋白的偶联将在培养类型中进行检测 2个细胞在丢失或被基质诱导恢复时， 表面活性物质的分泌功能，以及在2型细胞中衍生的 胎肺可以被诱导表达不同的特征- 体外实验。这些研究的完成应会扩大我们的 对表面活性物质分子调控机制的认识 2型细胞的分泌，并将为研究提供工具 肺泡上皮的成熟事件。

Alveolar type 2 pneumocytes synthesize and secrete lung surfactant in both constitutive and regulated modes. We have obtained evidence that GTP binding proteins participate in this pathway at two levels. First, several species of transductional G-proteins which couple surface receptors to effector molecules responsible for generating second messengers are present in type 2 cell membranes and cytosol. The presence of Gs, which couples the B- adrenergic receptor to adenylyl cyclase, had been implied by the earlier studies of others. We have obtained direct evidence for the presence of two species of Gi in addition to Gs. Further, we have shown that direct stimulation of the Gi proteins causes rapid and dramatic surfactant secretion. Preliminary evidence for the basis of stimulus-response coupling by this pathway is presented. Second, we have identified and partially purified three low molecular weight ras-related GTP binding proteins from lamellar bodies. Recent evidence from yeast genetics and mammalian in vitro complementation studies suggests that members of this family play key roles in the sorting and exocytosis of secretory vesicles. The goals of this project are to identify and characterize at a physiologic and molecular level the GTP binding proteins involved in stimulus-secretion coupling in type 2 cells. These goals will be pursued through three specific aims. 1) We will identify and quantitate the transductional G-protein species present in type 2 cells, establish the role of Gi proteins in mediating surfactant secretion and characterize the second messenger systems involved, and examine receptor mediated activation of this pathway. 2) We will purify low molecular weight GTP binding proteins from lamellar bodies, obtain partial protein sequence, clone cDNAs, produce anti- peptide antisera, and characterize the subcellular distribution and physiologic roles of these proteins. We will also determine if other low molecular weight GTP-binding proteins are present in non- lamellar body compartments of the type 2 cell. 3) Functional coupling of GTP binding proteins will be examined in cultured type 2 cells as they lose, or are induced by matrix to regain, their surfactant secretory function, and in type 2 cells derived from fetal lungs which can be induced to express features of differen- tiation in vitro. Completion of these studies should expand our knowledge of the molecular mechanisms regulating surfactant secretion by type 2 cells and will provide tools for studying maturational events in the alveolar epithelium.