STRUCTURE-FUNCTION ANALYSIS OF G PROTEIN SUBUNITS

我们的目标是确定独立委员会成员的职能 抑制性G蛋白家族。我们已经采用了遗传方法来 通过同源重组产生缺乏这两种基因的突变体 特定的基因产物，以确定它们在正常细胞中的作用 生理学。对这些突变体的分析，每个突变体都缺乏一个特定的阿尔法(I) G蛋白，应该能让我们确定这些蛋白的功能 G蛋白，就像细胞周期细胞揭示α/S的功能一样 含有G蛋白的。我们希望解决以下基本问题 和假设：1)受体-α的特异性是什么(I) 互动？2)α(I)-效应器的特异性是什么 互动？为了实现这些目标，我们将采取两种基因 方法，生产纯合子突变细胞系缺乏每个 α(I)亚基的克隆和转基因小鼠品系的建立 通过胚泡介导的转基因缺乏α(I)亚单位。 我们开发了一种生产纯合子突变株的新方法 细胞系。这种方法已经很容易地适用于许多基因 使我们能够制造出具有不止一个基因的细胞系 已停用。这些细胞系应该被证明是无价的工具 检验我们的假设并确定阿尔法(I)G-的具体作用 蛋白质。 使用这些细胞系，我们已经表达了异源受体。我们 将测试阿尔法(I)1、阿尔法(I)2和阿尔法(I)3到多个 受体包括D2-多巴胺受体、α2肾上腺素能受体和 5HT1a 5-羟色胺受体。我们已经确定了一个具体的要求 α(I)2在α2-肾上腺素能信号转导中的作用 受体对细胞内钙离子的反应，但不受基因敲除的影响 关于抑制cAMP积累的研究。我们会把这些研究扩展至 包括测试α(I)G蛋白在诱导有丝分裂中的作用 用蛙皮素。 胚胎干细胞是由内细胞衍生而来的培养细胞系 正常小鼠囊胚的质量。这些细胞能够形成所有 如果通过以下方式将小鼠的组织重新注入正常囊胚 微量注射。它们还能够在体外分化为 细胞类型的数量，包括跳动的心肌细胞，骨骼肌， 神经元、神经胶质细胞和造血细胞。我们将利用这一点 多潜能产生缺乏α(I)蛋白的ES细胞， 在体外将它们区分开来，然后测试它们与毒鼠强的偶联 膜片钳对心肌K+通道的受体作用 尽管关于α(I)亚基功能的许多问题是最好的 在培养的细胞系中回答一些问题需要生产 通过胚泡介导的转基因获得突变小鼠品系。在……里面 特别是，我们将研究α(I)2在生长和生长中的作用 肾上腺皮质和卵巢基质细胞的分化 Alpha(I)2被认为是一种原癌基因。

Our goal is to determine the function of the individual members of the inhibitory family of G-proteins. We have taken a genetic approach to produce mutants by homologous recombination which lack each of these specific gene products in order to determine their role in normal cell physiology. Analysis of these mutants, each lacking a specific alpha(i) G-protein, should enable us to determine the function of each of these G-proteins, just as the cyc-cells revealed the functions of alpha/s containing G-proteins. We wish to address the following basis questions and hypotheses: 1) What is the specificity of receptor-alpha(i) interactions? and 2) What is the specificity of alpha(i)-effector interactions? To accomplish these goals, we will take two genetic approaches, the production of homozygous mutant cell lines lacking each of the alpha(i) subunits, and the production of a transgenic mouse line lacking an alpha(i) subunit by blastocyst mediated transgenesis. We have developed a novel method for the production of homozygous mutant cell lines. This method has been readily adaptable for a number of genes and has allowed us to produce cell lines with more than one gene inactivated. These cell lines should prove to be invaluable tools to test our hypotheses and determine the specific roles of the alpha(i) G- proteins. Using these cells lines we have expressed heterologous receptors. We will test coupling of alpha(i)1, alpha(i)2 and alpha(i)3, to a number of receptors including D2-dopamine receptor, alpha2 adrenergic receptor and the 5HT1A serotonin receptor. We have identified a specific requirement for alpha(i)2 in the signal transduction from the alpha2-adrenergic receptor to intracellular Ca++ responses but no effect of the knockout on inhibition of cAMP accumulation. We will extend these studies to include testing the role of alpha(i) G-proteins on mitogenesis induced by bombesin. Embryonic stem cells are cultured cell line derived from the inner cell mass of normal mouse blastocyst. These cells are capable of forming all tissues of the mouse if reintroduced into a normal blastocyst by microinjection. They are also capable of in vitro differentiation to number of cell types including beating cardiocyte, skeletal muscle, neurons, glia, and hematopoietic cells. We will take advantage of this pluripotential to produce ES cells lacking the alpha(i) proteins, differentiate them in vitro, and then test coupling to muscarinic receptors to cardiac K+ channels by patch clamp. Although many questions about the function of alpha(i) subunits are best answered in cultured cell lines, some questions require the production of a mutant mouse line by blastocyst mediated transgenesis. In particular, we will study the role of alpha(i)2 on the growth and differentiation of adrenal cortical and ovarian stromal cells, where alpha(i)2 has been implicated as a proto-oncogene.