Agonist Activity at G Protein Coupled Receptors

描述(由申请人提供)：这项建议的目的是开发一种新的方法来估计G蛋白偶联受体的激动剂活性。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)，它相当于观察到的激动剂相对于标准激动剂的亲和力和内在效力的乘积。估计IRA所需的唯一数据是激动剂的浓度-反应曲线点。由于IRA值是激动剂-受体-G蛋白复合体的属性，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值在鉴定细胞系和分离组织中表达的重组受体的激动剂活性方面的有用性。研究计划估计在表达M2和M3以及β1和β2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结果进行比较，在天然组织中，以前的重组受体被认为介导了组织的反应。我们还计划评估在细胞系和豚鼠回肠中表达的M3受体激动剂的亲和力和相对有效性，并将这些估计的乘积(即亲和力x有效性)与IRA值进行比较。一些常用的研究激动剂活性的分析方法是在细胞匀浆上进行的，在这些细胞匀浆中，鸟嘌呤核苷酸的浓度往往与生理条件下保持的浓度不同。因此，我们计划研究GTP对腺苷环化酶测定中测定的激动剂在破碎细胞匀浆上的IRA值的影响。我们的工作假设是，GTP降低了观察到的激动剂的亲和力，但增加了它们的内在效力，导致在一定浓度范围内激动剂的IRA值没有变化。我们还计划比较在cAMP分析中估计的与Gs或Gi相关的受体上激动剂的IRA值，以及在受体与混杂G蛋白Galpha15和Galpha16共表达的细胞中用磷脂酰肌醇分析测得的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。这项建议中描述的简单的激动剂活性估计方法将极大地帮助发现新的治疗药物和研究G蛋白偶联受体在各种生理过程中的功能作用。我们提出的对Galpha15和Galpha16的研究具有非常重要的意义，因为这种G蛋白被广泛用作孤儿和已知受体的转导。

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.