Agonist Activity at G Protein Coupled Receptors

描述（由申请人提供）：本提案的目的是开发一种评估G蛋白偶联受体激动剂活性的新方法。我们的方法包括估计一个新的参数，称为激动剂的内在相对活性（IRA值），它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，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值在细胞系表达的重组受体及其在分离组织中的天然对应物上的激动剂活性的有效性。研究计划在表达M2和M3毒蕈碱和β 1和β 2肾上腺素受体的细胞系中估计激动剂的IRA值，并将这些估计值与在原组织中计算的估计值进行比较，原组织被认为是重组受体介导组织的反应。我们还计划估计激动剂在细胞系和豚鼠回肠中表达的M3毒蕈碱受体上观察到的亲和力和相对功效，并将这些估计值（即亲和力x功效）与IRA值进行比较。一些常用的研究激动剂活性的试验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的浓度不同。因此，我们计划研究GTP对破碎细胞匀浆上腺苷酸环化酶测定的激动剂IRA值的影响。我们的工作假设是，GTP降低了激动剂的观察亲和力，但增加了它们的内在功效，导致激动剂在GTP浓度范围内的IRA值没有变化。我们还计划比较在与G或Gi相关的受体上cAMP测定的激动剂的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化肌苷测定的受体的IRA值。我们的工作假设是，激动剂的IRA值可能（但不一定总是）独立于受体与之相互作用的G蛋白。本提案中描述的估计激动剂活性的简单方法将极大地有助于发现新的治疗剂和研究G蛋白偶联受体在各种生理过程中的功能作用。我们提出的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换能器具有重要意义。

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.