CONTROL OF SURFACTANT SECRETION BY GTP BINDING PROTEINS

肺泡2型细胞合成和分泌肺表面活性物质 无论是结构模式还是监管模式。 我们所获得 证明GTP结合蛋白参与这一途径， 两个层次。 首先，几种转导G蛋白 它将表面受体与效应分子结合 产生第二信使的基因存在于2型细胞 膜和胞质溶胶。 Gs的存在，其将B- 肾上腺素能受体腺苷酸环化酶，已暗示的 其他人的早期研究。 我们获得了直接证据， 除了G. 我们还 已经表明，直接刺激Gi蛋白会导致快速的 和显著的表面活性剂分泌。 初步证据表明， 提出了通过该途径进行刺激-反应耦合的依据。 第二，我们鉴定并部分纯化了三种低聚物， 板层组织中分子量ras相关GTP结合蛋白 尸体 酵母遗传学和哺乳动物体外实验的最新证据 互补研究表明这个家族的成员 在分泌囊泡的分选和胞吐中起关键作用。 的 该项目的目标是确定和表征 在生理和分子水平上， 在2型细胞的刺激分泌偶联中。 这些目标将 通过三个具体目标来实现。 1)我们将查明和 定量存在于2型中的转导G蛋白种类 细胞，建立Gi蛋白在介导表面活性剂 分泌和表征所涉及的第二信使系统， 并检测受体介导的该途径的激活。 2)我们 将从板层中纯化低分子量GTP结合蛋白， 体，获得部分蛋白序列，克隆cDNA，制备抗- 肽抗血清，并表征亚细胞分布， 这些蛋白质的生理作用。 我们还将确定， 其它低分子量GTP结合蛋白存在于非- 2型细胞的板层体隔室。 3)功能 GTP结合蛋白的偶联将在培养的类型中检查 2细胞，因为他们失去了，或被诱导的矩阵重新获得，其 表面活性剂分泌功能，并在2型细胞来源于 胎儿肺可以被诱导表达肺上皮细胞的特征， 体外实验 这些研究的完成将扩大我们的 调节表面活性剂的分子机理的知识 2型细胞的分泌，并将提供研究工具， 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.