Agonist Activity at G Protein Coupled Receptors

描述（由申请方提供）：本提案旨在开发一种新方法，用于估计G蛋白偶联受体的激动剂活性。我们的方法涉及估计一个新的参数，称为内在的相对活性的激动剂（伊拉值），这是相当于产品的观察到的亲和力和内在的效力的激动剂表示相对于一个标准的激动剂。估计伊拉所需的唯一数据是激动剂的浓度-反应曲线的点。由于伊拉值是激动剂-受体- G蛋白复合物的性质，可能不受下游信号转导机制的影响，因此伊拉值可用于比较不同反应系统中激动剂的活性。我们计划调查的有用性的伊拉值表征激动剂活性的重组受体表达的细胞系和他们的本地同行在分离的组织。计划进行研究，以估计表达M2和M3毒蕈碱受体以及β 1和β 2肾上腺素受体的细胞系中激动剂的伊拉值，并将这些估计值与天然组织中计算的值进行比较，其中认为前者的重组受体介导了组织的反应。我们还计划估计激动剂对在细胞系和豚鼠回肠中表达的M3毒蕈碱受体的观察到的亲和力和相对功效，并比较这些估计的乘积（即，亲和力×功效）与伊拉值。研究激动剂活性的一些常用测定法是在细胞匀浆中进行的，其中鸟嘌呤核苷酸的浓度通常不同于在生理条件下保持的浓度。因此，我们计划研究GTP对破碎细胞匀浆腺苷酸环化酶测定中确定的激动剂伊拉值的影响。我们的工作假设是，GTP降低了所观察到的亲和力的激动剂，但增加了其内在的功效，导致没有变化，在伊拉值的激动剂在一定范围内的GTP浓度。我们还计划比较的伊拉值的激动剂在cAMP测定的受体连接到Gs或Gi与那些在磷酸肌醇测定在细胞中的受体共表达与混杂的G蛋白Galpha 15和Galpha 16。我们的工作假设是，激动剂的伊拉值可能，但不一定总是，独立的G蛋白与受体相互作用。在这个建议中描述的激动剂活性的估计的简单方法，应大大有助于发现新的治疗药物和在各种生理过程中的G蛋白偶联受体的功能作用的研究。我们提出的Galpha 15和Galpha 16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换器是非常重要的。

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.