Molecular Characterization of GPR35 and GPR55, Putative Cannabinoid Receptors

描述(申请人提供)：大量的生理过程由内源性大麻素控制。这些效应大多归因于大麻素CB1或CB2受体的作用。然而，有一些效应显然不是CB1或CB2介导的，也可能不是受体介导的，但也有足够的证据表明香草素受体(TRPV1)参与了大麻素的效应，至少还有另外两种大麻素受体亚型到目前为止只被药理学定义。最近，两个孤儿G蛋白偶联受体(GPCRs)GPR35和GPR55被认为是大麻素受体，据报道，相当广泛的大麻素配体在每个受体上都显示出亲和力/功效。我们已经克隆并表达了GPR35和GPR55，我们的初步研究证实它们可以被多种大麻类化合物激活。在这里提出的工作中，我们计划通过配体结合和功能研究来表征这两个GPCRs。最近开发的GPR35和GPR55处于非激活和激活状态的计算机模型将用于指导每个受体的突变研究。这些模型是由我们对大麻素CB1和CB2受体的广泛建模和突变经验提供信息的。突变研究的目标不仅是确定与配基识别有关的残基，还包括那些对受体激活重要的残基。在GPR35和GPR55配基识别研究中，将使用精心挑选的化合物来测试具有特定配基功能基团的特定氨基酸的参与。体外突变研究的结果将被用来改进计算机受体模型，这样在任何给定的时间，这些模型都能反映该领域的当前知识状态。由于到目前为止，对GPR35和GPR55的研究还没有发现针对这两种受体的高亲和力拮抗剂，而且这种拮抗剂将是研究这两种受体的非常有价值的工具，因此拟议工作的另一个目标是为每种受体亚型设计拮抗剂。这些化合物将在瑞吉奥设计，在三角研究所(塞尔茨曼)合成，并在CPMCRI(ABOOD)进行评估。这项工作产生的高亲和力拮抗剂将被放射性标记并提供给科学界。确定GPR35的分布以及GPR35和GPR55的信号转导途径将有助于确定它们的生理和病理生理作用。

DESCRIPTION (provided by applicant): A large number of physiological processes are controlled by the endogenous cannabinoids. Most of these effects have been attributed to action at either the cannabinoid CB1 or CB2 receptors. Yet there are effects that clearly are not CB1- or CB2-mediated which may not be receptor mediated, but there is also sufficient evidence to suggest the involvement of the vanilloid receptor (TRPV1) in cannabinoid effects and at least two other cannabinoid receptor subtypes defined only pharmacologically until now. Very recently, two orphan G-protein coupled receptors (GPCRs), GPR35 and GPR55, have been suggested to be cannabinoid receptors, with a fairly wide range of cannabinoid ligands reported to display affinity/efficacy at each. We have cloned and expressed both GPR35 and GPR55 and our preliminary studies confirm that they are activated by multiple cannabinoid compounds. In work proposed here, we plan to characterize these two GPCRs through ligand binding and functional studies. Recently developed computer models of GPR35 and GPR55 in their inactive and activated states will be used to guide mutation studies of each receptor. These models are informed by our extensive modeling and mutation experience with the cannabinoid CB1 and CB2 receptors. The goal of mutation studies will be not only to identify residues involved in ligand recognition, but also those residues important for receptor activation. In GPR35 and GPR55 ligand recognition studies, the involvement of specific amino acids with specific ligand functional groups will be tested using carefully chosen compounds. Results of mutation studies in vitro will be used to refine computer receptor models, such that at any given time, these models reflect the current state of knowledge in the field. Because work thus far on GPR35 and GPR55 has not identified a high affinity antagonist for either receptor and because such antagonists would be very valuable tools for studying these two receptors, another goal of the proposed work will be to design antagonists for each receptor sub-type. These compounds will be designed at UNCG (Reggio), synthesized at Research Triangle Institute (Seltzman), and evaluated at CPMCRI (Abood). High affinity antagonists that emerge from this work will be radiolabeled and made available to the scientific community. Determining the distribution of GPR35 and signal transduction pathways of GPR35 and GPR55 will help define their physiological and pathophysiological roles.