SF9 CELL STUDY OF 5 HT1A RECEPTOR G PROTEIN COUPLING

5 HT1A 受体 G 蛋白偶联的 SF9 细胞研究

• 批准号: 6111553
• 负责人: David Randell Manning
• 金额: $ 17.61万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6111553
• 关键词: G protein; Sf9 cell line; affinity labeling; immunoprecipitation; model design /development; neurotransmitter agonist; neurotransmitter antagonist; receptor binding; receptor coupling; receptor sensitivity; serotonin receptor; western blottings

The objective of the proposed research is to exploit the insect (Sf9 cell) baculovirus expression system as a model for characterizing 5-HT(1A) receptor G protein interactions. Our first goal is to develop Sf9 cells expressing the 5-HT(1A) receptor +/- G protein subunits as a model for defining full agonists, partial agonists, inverse agonists, and antagonists. Sf9 cells are uniquely suited to this kind of analysis, as they permit measurements of "efficacy" based on receptor G protein communication alone. We propose to classify ligands according to i) whether a G protein such as G(i) alters the affinity of the 5-HT(1A) receptor for a partial ligand (thus classifying the ligand as an agonist), and, if so, whether the affinity is increased (full or partial agonist) or decreased (inverse agonist), ii) whether wild-type and "constitutively active" 5-HT(1A) receptors differ in their affinities for a particular ligand (permitting discrimination of agonists from neutral antagonists, and, among agonists, full, partial, and inverse agonists), and iii) whether a particular ligand stimulates (full or partial agonist), suppresses (inverse agonist), or has no effect on (antagonist) G protein activity. Work will also be carried out using the system to define irreversible and photoaffinity ligands. Our second goal is to identify G proteins that interact with the 5-HT(1A) receptor. We will turn our attention to those G proteins whose identities have only recently emerged and/or whose functions are sufficiently unclear as to hamper other forms of analysis. These G proteins include sGi2, G12, G13, G14, and G15/16. We will additionally analyze beta and gamma subunits as determinants of specificity. Our third goal is to develop and/or validate techniques used to map receptor G protein communication in mammalian tissues. The advantage of the Sf9 Cell/Baculovirus model is that the interactions between 5-HT1A receptors and G proteins can be tested one G protein at a time, with the G protein unambiguously defined by the combination of subunits introduced. We will concentrate our efforts on two techniques - receptor G protein co-immunoprecipitation and agonist-promoted [35S]GTPgammaS-binding-comparing the results achieved with pharmacological indices of coupling.

本研究的目的是利用昆虫（Sf 9细胞） 杆状病毒表达系统作为表征5-HT（1A）的模型 受体G蛋白相互作用。 我们的第一个目标是开发Sf 9细胞 表达5-HT（1A）受体+/- G蛋白亚基作为模型， 定义完全激动剂、部分激动剂、反向激动剂，和 对手。 sf 9细胞是唯一适合这种分析，因为 它们允许基于受体G蛋白测量“功效”， 单独沟通。 我们建议根据i）对配体进行分类 G蛋白如G（i）是否改变5-HT（1A）的亲和力 部分配体的受体（因此将配体分类为激动剂）， 以及如果是，亲和力是否增加（完全或部分激动剂）或 减少的（反向激动剂），ii）无论是野生型还是“组成型 活性”5-HT（1A）受体在它们对特定的 配体（允许区分激动剂和中性拮抗剂， 以及，在激动剂中，完全、部分和反向激动剂），和iii） 无论特定配体刺激（完全或部分激动剂）， 抑制（反向激动剂），或对（拮抗剂）G蛋白没有影响 活动 还将利用该系统开展工作， 不可逆和光亲和配体。 我们的第二个目标是确定G 与5-HT（1A）受体相互作用的蛋白质。 我们将把我们的 注意那些G蛋白，它们的身份只是最近才出现的 和/或其功能不够明确，妨碍了其他形式的 的分析。 这些G蛋白包括sGi 2、G12、G13、G14和G15/16。 我们还将分析β和γ亚基作为 的特异性 我们的第三个目标是开发和/或验证所使用的技术 绘制哺乳动物组织中受体G蛋白通讯的图谱。 的 Sf 9细胞/杆状病毒模型的优点是， 5-HT 1A受体和G蛋白之间的关系可以在一个G蛋白上进行测试。 时间，与G蛋白明确定义的组合， 子单位介绍。 我们将集中精力在两项技术上- 受体G蛋白免疫共沉淀和激动剂促进 [35 S] GTP γ S-结合-比较药理学 耦合指数