STRUCTURE-FUNCTION ANALYSIS OF G PROTEIN SUBUNITS

我们的目标是确定每个成员的功能， G蛋白抑制家族。 我们采用了基因方法 通过同源重组产生缺乏这些基因的突变体， 特异性基因产物，以确定它们在正常细胞中的作用 physiology. 对这些突变体的分析，每个突变体都缺乏特定的α（i） G蛋白，应该能让我们确定每一种蛋白的功能 G蛋白，正如细胞周期细胞揭示了α/S的功能一样， 含有G蛋白 我们希望解决以下基本问题 1）受体α（i）的特异性是什么？ 互动？和2）α（i）-效应子的特异性是什么 互动？ 为了实现这些目标，我们将采取两种遗传学方法， 方法，生产缺乏每个纯合突变细胞系， 的α（i）亚基，以及转基因小鼠品系的产生， 通过胚泡介导的转基因缺乏α（I）亚单位。 我们已经开发了一种新的方法生产纯合突变体 细胞系 这种方法已经很容易地适用于许多基因 并使我们能够生产出具有多个基因的细胞系 灭活 这些细胞系应该被证明是无价的工具， 测试我们的假设，并确定alpha（i）G的具体作用- proteins. 使用这些细胞系，我们表达了异源受体。 我们 将测试α（i）1、α（i）2和α（i）3与多个 受体，包括D2-多巴胺受体、α 2肾上腺素能受体和 5 HT 1A血清素受体 我们已经确定了一个具体的要求 对于α（i）2在从α 2-肾上腺素能神经元的信号转导中的作用， 受体对细胞内Ca++的反应，但敲除无影响 抑制cAMP积累。 我们将把这些研究扩展到 包括测试α（i）G蛋白对诱导的有丝分裂的作用， 关于Bombesin 胚胎干细胞是由内细胞分化而来的培养细胞系 正常小鼠囊胚的质量。 这些细胞能够形成 如果通过以下方法将小鼠的组织重新引入正常胚泡， 显微注射 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、神经胶质和造血细胞。 我们将利用这一点 多能性以产生缺乏α（i）蛋白的ES细胞， 在体外将它们分化，然后测试与毒蕈碱的偶联， 通过膜片钳技术检测心脏K+受体与心脏K+通道的相互作用。 尽管关于alpha（i）亚基功能的许多问题最好 在培养的细胞系中回答，有些问题需要生产 通过囊胚介导的转基因获得突变小鼠品系。 在 特别是，我们将研究α（i）2对生长的作用， 肾上腺皮质和卵巢基质细胞的分化，其中 α（i）2被认为是原癌基因。

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.