TRANSMEMBRANE SIGNALING VIA RECEPTORS AND G-PROTEINS

战略提出了阐明机制，家庭 膜相关蛋白，G蛋白，介导的调节， 细胞外激素的细胞内过程。 这个蛋白质家族 包括Gs和Gi，介导刺激的调节蛋白， 腺苷酸环化酶和调节蛋白Gt（转导蛋白）抑制 的视觉系统。 第四种蛋白质，Go，在体内大量存在， 脑组织，并与毒蕈碱受体相互作用; Go的功能是 未知 毒蕈碱受体已被选为受体成分 用于复溶研究。 因此，所有组件都将被导出 牛的CNS。 G蛋白的纯化和表征将是连续的 目标. 这将包括完善现行程序和发展 新的方法来纯化单个亚基。 表征 包括试图使Go的α亚基结晶， 在不存在去污剂的情况下G蛋白的溶解度，以及各种 活动测量。 这些测量将集中在Go蛋白， 包括评估核苷酸结合和GT3活性的作用 在蛋白质的激活中。 G蛋白与 磷脂囊泡将试图评估膜的作用， G蛋白的作用。 Alphao和Alphai的可逆缔合 将利用含有BetaGamma的囊泡来研究 活化中的亚基解离。 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，并作为探针，以确定潜在的 监管目标。 Go的特定antigodies将用于获得 这种蛋白质在神经组织中的细胞和细胞内位置。 用于有效重建受体和G蛋白的方法， 磷脂囊泡将是最初的优先事项。 的特异性 受体与不同G蛋白的相互作用以及 将研究各种亚单位。 毒蕈碱的纯化 受体将利用已公布的技术以及新技术， 建议使用生物素化拮抗剂或G蛋白作为亲和力 受体的探针 长期目标是重建纯化的组分（受体， G蛋白和调节酶）。 这样， 这一共同信号系统的机制可以从激素 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.