New tools for studying receptor dimers

摘要 代谢型谷氨酸受体(MGluRs)是C类G蛋白偶联受体，具有二聚体的功能。 虽然已知mGluR形成同源二聚体，但最近的研究表明，它们也可以形成异二聚体， 但不是杂乱无章的。因为mGluRs在大脑中广泛表达，调节兴奋性和 可塑性，它们已成为各种病理疾病的候选药物靶点。然而到目前为止， 临床前数据产生的兴奋并未导致临床上的mGluR靶向治疗，尽管 以这些受体为靶标的大量可用配体具有良好的选择性。我们最近研究mGluR2/4的工作 杂二聚体提供了一个可能的解释：当靶向同二聚体时，配体是非常有效的 当同一受体与另一种mGluR以异二聚体的形式表达时，受体通常是无效的。 更复杂的是，在mGluR2/4异二聚体中观察到的这些药理反应的变化 并不适用于所有的mGluR异二聚体，甚至不适用于所有含有异二聚体的mGluR2。因此，要 要了解mGluR配体在大脑中的功能，我们必须检查药物反应 每一对可能的异源二聚体的分离。但这很复杂，因为每个mGluR也可以形成 同源二聚体，所以任何一对表达的mGluR都有未知的同源和异源二聚体倾向。 为了解决这个问题，我们设计了一种新颖的使用电流变组合的二聚体合成控制系统 保留序列与正交的、分裂的内含子、自切除的蛋白质序列配对，这将允许 表达已知组成的接近野生型mGluR二聚体的纯群体。这个系统将是 不仅在定义每个二聚体对的药理学行为方面有价值，而且还通过提供更广泛的 更准确地描绘mGluR靶向配体的影响，作为广泛范围的 药物筛查。此外，该策略还为其他二聚化膜蛋白的研究提供了模板。 为此，我们将追求以下具体目标：1-确定我们的 新的真核分裂内含子系统，以及2-产生并检测每个mGluR与分裂内含子的结合 内含素系统。

Abstract Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors that function as dimers. While mGluRs are known to form homodimers, more recent work has shown that they can also heterodimerize, but not promiscuously. Because mGluRs exhibit widespread expression in the brain and regulate excitability and plasticity, they have become candidates as druggable targets for a variety of pathologies. To date however, excitement generated by preclinical data has not resulted in mGluR-targeting therapies in the clinic, despite a wealth of available ligands with good selectivity targeting these receptors. Our recent work examining mGluR2/4 heterodimers provides a possible explanation: ligands that are highly efficacious when targeting homodimeric receptors are often without effect when the same receptor is expressed as a heterodimer with another mGluR. Further complicating matters, these changes in pharmacological responses observed in mGluR2/4 heterodimers are not generalizable to all mGluR heterodimers, or even all mGluR2 containing heterodimers. Thus, to understand how any mGluR ligand will function in the brain, we must examine the pharmacological responses of each possible heterodimer pair in isolation. But this is complicated because every mGluR can also form homodimers, so any pair of expressed mGluR will have an unknown propensity to homo- and heterodimerize. To solve this problem, we have designed a novel dimer composition control system using a combination of ER retention sequences paired with orthogonal, split inteins, self-excising protein sequences, that will allow expression of pure populations of nearly wild type mGluR dimers of known composition. This system will be valuable not only in defining the pharmacological behavior of each dimer pair, but also by providing a broader and more accurate picture of the effects of mGluR targeting ligands by serving as a platform for broadly scoped drug screening. Further, this strategy provides a template for the study of other dimerizing membrane proteins. To this end, we will pursue the following Specific Aims: 1- Determine the performance and limitations of our novel eukaryotic split-intein system, and 2- Generate and test each mGluR combination with the split- intein system.