STRUCTURE AND FUNCTION OF SIGNAL-TRANSDUCING G-PROTEINS

信号转导 G 蛋白的结构和功能

• 批准号: 5203780
• 负责人: J NORTHUP
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5203780
• 关键词: Baculoviridae; G protein; biological signal transduction; bombesin; conformation; isoprenoid; neuropeptide receptor; protein reconstitution; protein structure function; receptor coupling; receptor expression; rhodopsin; serotonin receptor

In the past year we have characterized and extended our new in situ receptor-G-protein assay systems to increase their utility for a diverse family of receptor structures. The retinal activation of apo-opsin has been examined as a molecular model for ligand-regulated G-protein coupled receptors; and the relationships between G-protein alpha and betagamma subunit and retinal inreactions and rhodopsin conformations tested. these experiments show that as seen for bona fide ligand-regulated receptors retinal activation of apo-opsin displays two affinity states. However the high affinity interaction of opsin with retinal appears to be controlled betagamma rather than a subunit interaction. We have exploited the nucleotide regulation of these interactions to devise a highly specific sensitive biochemical assay for receptor-activation of G-proteins and confirmed its utility for bovine and squid rhodpsins and 5HT2c receptors. We have extended the expression of G-protein-coupled receptors using baculorvirus to the bombesin receptor family and initiated examination of the properties of the uncoupled and G-protein- coupled GRP receptor using our in situ reconstitution procedures. Lastly, as an initial test of the structural basis for Gbetagamma functional differences we have constructed mutations of the Ggamma2 gene to encode for differing isoprenoid modification. These studies show that the betagamma interaction with alpha or receptor is dependent upon isoprenylation, but that the specific isoprenoid modification of the gamma2 makes no difference to the alpha interaction and only modestly alters receptor interaction. These data support the hypothesis that the diverse betagamma protein structures encode receptor specific contacts.

在过去的一年里，我们描述并扩展了我们新的原位 受体-G-蛋白测定系统，以提高其在多种应用中的实用性 受体结构家族。 脱辅基视蛋白的视网膜激活 作为配体调节的 G 蛋白偶联的分子模型进行了检查 受体；以及G蛋白α和βγ之间的关系 测试了亚基和视网膜反应以及视紫红质构象。 这些实验表明，正如所见，真正的配体调节 脱辅基视蛋白的受体视网膜激活表现出两种亲和状态。 然而，视蛋白与视网膜的高亲和力相互作用似乎 受βγγ控制而不是亚基相互作用。 我们有 利用这些相互作用的核苷酸调节来设计 受体激活的高度特异性灵敏的生化测定 G 蛋白并证实了其对于牛和鱿鱼视紫红质的效用 5HT2c受体。 我们扩展了 G 蛋白偶联的表达 使用杆状病毒对铃蟾肽受体家族的受体和 开始检查未偶联和 G 蛋白的特性 使用我们的原位重建程序偶联 GRP 受体。 最后，作为 Gbetagamma 结构基础的初步测试 功能差异 我们构建了 Ggamma2 基因的突变 编码不同的类异戊二烯修饰。 这些研究表明 βγ 与 α 或受体的相互作用取决于 异戊二烯化，但特定的类异戊二烯修饰 gamma2 对 alpha 相互作用没有影响，并且影响不大 改变受体相互作用。 这些数据支持以下假设： 不同的βγ蛋白结构编码受体特异性接触。