Agonist Activity at G Protein Coupled Receptors

G 蛋白偶联受体的激动剂活性

• 批准号: 6869790
• 负责人: Fred J Ehlert
• 金额: $ 23.57万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6869790
• 关键词: CHO cells; G protein; adenylate cyclase; biological signal transduction; cell line; cell surface receptors; computational biology; guanosine triphosphate; guinea pigs; method development; receptor coupling; receptor expression; smooth muscle; statistics /biometry; transfection

DESCRIPTION (provided by applicant): The aim of this proposal is to develop a novel method for the estimation of agonist activity at G protein coupled receptors. Our method involves estimating a new parameter termed the intrinsic relative activity of the agonist (IRA value), which is equivalent to the product of the observed affinity and intrinsic efficacy of an agonist expressed relative to that of a standard agonist. The only data required for estimation of IRA are the points of the concentration-response curve of the agonist. Since the IRA value is a property of the agonist-receptor- G protein complex and is probably unaffected by downstream signal transduction mechanisms, the IRA value is useful for comparing the activity of agonists across different response systems. We plan to investigate the usefulness of the IRA value for characterizing agonist activity at recombinant receptors expressed in cell lines and their native counterparts in isolated tissues. Studies are planned to estimate the IRA values of agonists in cell lines expressing M2 and M3 muscarinic and Beta 1 and Beta2 adrenoceptors and to compare these estimates with those calculated in native tissues where the former recombinant receptors are thought to mediate the responses of the tissues. We also plan to estimate both the observed affinity and relative efficacy of agonists at M3 muscarinic receptors expressed in cell lines and guinea pig ileum and to compare the product of these estimates (i.e., affinity x efficacy) with the IRA value. Some commonly used assays for investigating agonist activity are carried out on cellular homogenates where the concentration of guanine nucleotides often differs from that maintained under physiological conditions. Consequently, we plan to investigate the effects of GTP on the IRA values of agonists determined in adenylyl cyclase assays on broken cell homogenates. Our working hypothesis is that GTP reduces the observed affinity of agonists but increases their intrinsic efficacy, resulting in no change, in the IRA value of the agonist over a range of concentrations of GTP. We also plan to compare the IRA values of agonists estimated in cAMP assays on receptors linked to Gs or Gi with those measured in phosphoinositide assays in cells where the receptors are coexpressed with the promiscuous G proteins Galpha15 and Galpha16. Our working hypothesis is that the IRA values of agonists are probably, but not necessarily always, independent of the G protein with which the receptor interacts. The simple method for estimation of agonist activity described in this proposal should greatly aid in the discovery of novel therapeutic agents and in the study of the functional role of G protein coupled receptors in various physiological processes. Our proposed investigation of Galpha15 and Galpha16 is highly significant with regard to the widespread use of this G protein as a transducer for both orphan and known receptors.

描述（由申请人提供）：本提案的目的是开发一种用于估计 G 蛋白偶联受体激动剂活性的新方法。我们的方法涉及估计一个称为激动剂内在相对活性（IRA 值）的新参数，该参数相当于观察到的亲和力和激动剂相对于标准激动剂表达的内在功效的乘积。估计 IRA 所需的唯一数据是激动剂浓度-反应曲线上的点。由于 IRA 值是激动剂-受体-G 蛋白复合物的特性，并且可能不受下游信号转导机制的影响，因此 IRA 值可用于比较不同反应系统中激动剂的活性。我们计划研究 IRA 值对于表征细胞系中表达的重组受体及其分离组织中的天然对应物的激动剂活性的有用性。计划研究估计表达 M2 和 M3 毒蕈碱以及 Beta 1 和 Beta2 肾上腺素受体的细胞系中激动剂的 IRA 值，并将这些估计值与在天然组织中计算的值进行比较，在天然组织中，前重组受体被认为介导组织的反应。我们还计划估计观察到的激动剂对细胞系和豚鼠回肠中表达的 M3 毒蕈碱受体的亲和力和相对功效，并将这些估计的乘积（即亲和力 x 功效）与 IRA 值进行比较。一些用于研究激动剂活性的常用测定是在细胞匀浆上进行的，其中鸟嘌呤核苷酸的浓度通常与生理条件下维持的浓度不同。因此，我们计划研究 GTP 对破碎细胞匀浆的腺苷酸环化酶测定中测定的激动剂 IRA 值的影响。我们的工作假设是，GTP 降低了观察到的激动剂亲和力，但增加了其内在功效，导致激动剂在一定浓度的 GTP 范围内的 IRA 值没有变化。我们还计划将在与 Gs 或 Gi 相关的受体的 cAMP 测定中估计的激动剂 IRA 值与在受体与混杂的 G 蛋白 Galpha15 和 Galpha16 共表达的细胞中磷酸肌醇测定中测量的 IRA 值进行比较。我们的工作假设是激动剂的 IRA 值可能但不一定总是独立于与受体相互作用的 G 蛋白。该提案中描述的估计激动剂活性的简单方法应该极大地有助于新型治疗剂的发现以及G蛋白偶联受体在各种生理过程中的功能作用的研究。我们提出的对 Galpha15 和 Galpha16 的研究对于广泛使用这种 G 蛋白作为孤儿和已知受体的转导器具有非常重要的意义。