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: 6204856
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-09-15 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6204856
• 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的Sf9细胞。