TRANSMEMBRANE SIGNALING VIA RECEPTORS AND G-PROTEINS

人们提出了一些策略，用于阐明一个家庭 膜相关蛋白，G蛋白，介导调节 由细胞外激素引起的细胞内过程。这一蛋白质家族 包括Gs和Gi，这是一种调节蛋白，介导刺激和 抑制腺苷环化酶和调节蛋白GT(转导蛋白) 视觉系统的一部分。第四种蛋白质GO在体内含量很高 并与M受体相互作用；GO的功能是 未知。M受体被选为受体成分。 用于重建研究。因此，将派生出所有组件 来自牛中枢神经系统。 G-蛋白的纯化和鉴定将是连续的 目标。这将包括完善当前的程序和发展。 提纯单个亚基的新方法。表征 包括尝试结晶围棋的阿尔法亚单位，对 G-蛋白在不含洗涤剂的情况下的溶解性 活动测量。这些测量将集中在GO蛋白质和 包括评估核苷酸结合和GTPase活性的作用 在蛋白质的激活中。G蛋白与人类免疫功能的关系 磷脂小泡将试图评估膜在 G蛋白的作用。阿尔卑斯和阿尔菲的可逆组合 含有BetaGamma的囊泡将被利用来研究 亚基在激活时解离。 围棋的功能将通过尝试使用纯化的围棋来探索 调节PI代谢和离子通量，并作为识别潜力的探针 监管的目标。特定的抗滴剂将被用来获得 这种蛋白质在神经组织中的细胞和细胞内的位置。 受体和G-蛋白的高效重组方法 磷脂囊泡将是最初的优先事项。它的特殊性 受体与不同G蛋白的相互作用及其要求 将对各种亚基进行研究。毒扁豆碱的提纯 接受者将利用已发表的技术以及新技术 建议使用生物素化拮抗剂或G蛋白作为亲和力 探查感受器。 长期目标是重建纯化的成分(受体， 磷脂小泡中的G蛋白和调节酶)。就这样， 这一共同的信号系统的作用机制可以从激素角度进行研究。 在一个明确的环境中发挥作用。

Strategies are proposed for the elucidation of mechanisms by which a family of membrane-associated proteins, G-proteins, mediate regulation of intracellular processes by extracellular hormones. This family of proteins includes Gs and Gi, the regulatory proteins that mediate stimulation and inhibition of adenylate cyclase, and Gt (transducin), a regulatory protein of the visual system. A fourth protein, Go, is found in high quantity in brain tissue and interacts with muscarinic receptors; the function of Go is unknown. The muscarinic receptor has been chosen as the receptor component for reconstitution studies. Thus, all of the components will be derived from bovine CNS. Purification and characterization of the G-proteins will be continuous goals. This will include refinement of current procedures and development of new methods for purification of individual subunits. Characterization includes attempts to crystallize the Alpha subunit of Go, studies of the solubility of the G-proteins in the absence of detergent, and a variety of activity measurements. These measurements will focus on the Go protein and include assessment of the role of nucleotide binding and GTPase activities in activation of the proteins. The association of the G-proteins with phospholipid vesicles will attempt to assess the role of the membrane in the action of G-proteins. The reversible association of Alphao and Alphai with vesicles that contain BetaGamma will be exploited to study the role of subunit dissociation in activation. The function of Go will be explored by attempts to use purified Go to regulate PI metabolism and ion fluxes, and as a probe to identify potential targets of regulation. Specific antigodies to Go will be used to obtain the cellular and intracellular location of this protein in neural tissue. Methods for efficient reconstitution of receptors and G-proteins in phospholipid vesicles will be an initial priority. The specificity of interaction of the receptor with different G-proteins and the requirement for various subunits will be studied. Purification of the muscarinic receptor will utilize published techniques as well as new techniques that propose to use a biotinylated-antagonist or the G-proteins as affinity probes for the receptor. The long-term goal is to reconstitute purified components (receptors, G-proteins, and regulated enzymes) in phospholipid vesicles. In this way, the mechanisms of this common signaling system can be studied from hormone to effect in a well-defined milieu.