ACTION OF HORMONES ON ADENYLYL CYCLASE SYSTEMS

异三聚体 G 蛋白（亚基组成 α.Beta/γ）介导 一大类受体通过其激活作用而产生的影响 或对效应器系统的失活作用。 过去几年已经看到 阐明 16 种 G 蛋白中 14 种的主要功能 现在已知的α亚基，以及β/γ二聚体 受体下 GTP 激活 G 蛋白而形成 影响。 最近的研究表明三聚体 G 蛋白参与其中 不仅调节腺苷酸环化酶 (AC)、磷脂酶 （PL）、磷酸二酯酶和离子通道，而且还存在于诸如 作为从内质网和高尔基体网络出芽的囊泡，以及 内体酸化。 信号转导具有更广泛的作用 表示为 G 蛋白。 Galpha 亚基突变导致 在赘生物和肿瘤中发现了组成型激活，并且它 假设某些α亚基可能是原癌基因。 分子克隆和随后的预测初级阐明 AC 的结构导致了很大程度上分子和 功能多样性（8 个基因，产品可根据 对 Ca2、钙调蛋白、β/γ 的反应，并可能被激活 Gi/alpha 的）。 单个细胞中最多可表达 5 个 AC 亚型： 广泛用于研究调节的垂体肿瘤细胞 腺苷酸环化酶、磷脂酶、离子通道和激素分泌。 在上学期我们在实验室建立了这项技术 使胚胎干细胞中的基因失活或突变所需的 通过注射到基因组发生改变的动物胚泡中产生。 应用这种技术敲除我们获得的 Gi2 的 alpha 亚基 新信息不仅涉及其在调节荷尔蒙方面的可疑作用 抑制腺苷酸环化酶，而且还发现小鼠缺乏 αi2 中的 T 细胞发育和功能异常，并发展为 类似于溃疡性结肠炎的炎症性肠病。 无论是这些 现象之间是否存在因果关系尚不清楚。 本申请提出的研究完全集中于生成新颖的 含有 alpha/0、alpha/i1、alpha i3 基因的小鼠品系 失活并且其中α/s和α/13和α/i2是 突变激活。 通过育种，我们将结合一种或多种 这些突变。 G 蛋白的其他修饰或失活 将考虑并执行亚单位（例如 alpha/o1 与 alpha/o2） 取决于结果和进展速度。 希望这些研究能够 1. 测试 G 蛋白的作用 在发育过程以及细胞功能中尚未 被认为是 G 蛋白调节的目标； 2. 严格测试 正常小鼠背景下 Galpha 的原癌基因潜力； 3.测试α亚基的可能功能，其中没有 函数到目前为止是已知的。

Heterotrimeric G proteins (subunit composition alpha.Beta/gamma) mediate the effects of a large family of receptors by virtue of their activating or deactivating effects on effector systems. The last years have seen the elucidation of the primary functions of 14 out of the 16 G protein alpha subunits that are now known, as well as for Beta/gamma dimers that form as a result of G proteins activation by GTP under the receptor influence. Recent studies have shown involvement of trimeric G proteins not only in regulation of adenylyl cyclases (AC's), phospholipases (PL's), phosphodiesterases and ion channels, but also in processes such as vesicle budding from endoplasmic reticulum and Golgi networks, and in endosome acidification. Broader roles that mere signal transduction are indicated for G proteins. Mutations of the Galpha subunits that cause constitutive activation have been found in neoplasms and tumors and it has been hypothesized that some alpha subunits may be proto-oncogenes. The molecular cloning and ensuing elucidation of the predicted primary structure of AC led to the discovery of a large degree of molecular and functional diversity (8 genes with products sub-classifiable on the basis of response to Ca2+, calmodulin, Beta/gamma, and possibly activated Gi/alpha's). Up to 5 AC subtypes may be expressed in a single cell as seen with a pituitary tumor cell widely used to study regulation of adenylyl cyclase, phospholipase, ion channel and hormone secretion. During this last term we established in out laboratory the technology required to inactivate or mutate genes in embryonic stem cells and generate via injection into blastocysts animals with an altered genome. Applying this technique to knock out the alpha subunit of Gi2 we gained new information not only about its suspected role in mediating hormonal inhibition of adenylyl cyclase, but also discovered that mice deficient in alphai2 have abnormal T cell-development and function and develop an inflammatory bowel disease resembling ulcerative colitis. Whether these phenomena are causally related is not known. Research proposed in this application focusses wholly on generating novel mouse strains in which genes for alpha/0, alpha/i1, alpha i3 are inactivated and in which alpha/s and alpha/13 and alpha /i2 are mutationally activated. Through breeding, we will combine one or more of these mutations. Other modifications or inactivation of G protein subunits (e.g. alpha/o1 vs. alpha/o2) will be considered and performed depending on outcome and the speed of progress. It is hoped that these studies will 1. test for role(s) of G proteins in developmental processes as well as in cellular functions not yet thought of as targets of G protein regulation; 2. critically test the proto-oncogene potential of Galpha's in the background of a normal mouse; and 3. test for possible functions of alpha subunits for which no function is thus far known.