SF9 CELL STUDY OF 5 HT1A RECEPTOR G PROTEIN COUPLING

这项拟议的研究的目的是开发这种昆虫(Sf9细胞)。 杆状病毒表达系统作为表征5-羟色胺(1A)的模型 受体G蛋白相互作用。我们的第一个目标是开发Sf9细胞 5-羟色胺(1A)受体+/-G蛋白亚基的表达 定义完全激动剂、部分激动剂、反向激动剂和 对抗者。Sf9细胞特别适合于这种分析，因为 它们允许测量基于受体G蛋白的“疗效” 单独交流。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-羟色胺(1A)的亲和力 部分配体的受体(从而将该配体归类为激动剂)， 如果是，亲和力是否增加(全部或部分激动剂)或 减少(反向激动剂)，ii)是否为野生型 活性“5-羟色胺(1A)受体对特定受体的亲和力不同 配体(允许区分激动剂和中性拮抗剂， 以及在激动剂中，完全激动剂、部分激动剂和反向激动剂)，以及 特定的配体是否刺激(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，以确定 不可逆光亲和性配体。我们的第二个目标是确定G 与5-羟色胺(1A)受体相互作用的蛋白质。我们将把我们的 注意那些最近才出现身份的G蛋白 和/或其功能不明确到足以阻碍其他形式 分析的能力。这些G蛋白包括SGI2、G12、G13、G14和G15/16。 我们还将分析β亚基和伽马亚基作为决定因素 专一性。我们的第三个目标是开发和/或验证使用的技术 绘制哺乳动物组织中受体G蛋白的通讯图谱。这个 Sf9细胞/杆状病毒模型的优点是 在5-HT1a受体和G蛋白之间可以一次测试一个G蛋白 时间，与G蛋白明确定义的组合 引入了亚单位。我们将集中力量研究两种技术- 受体G蛋白免疫共沉淀与激动剂促进 [35S]GTP-GammaS结合--与药理学结果的比较 耦合指数。

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.