A novel system for controlling dimeric receptor composition to discover unique heterodimer pharmacology

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，并调节兴奋性 和可塑性，它们已经成为各种病理疾病的候选药物靶点。到目前为止 然而，临床前数据产生的兴奋并没有导致临床上的mGluR靶向治疗， 尽管有丰富的可用配体具有良好的选择性，但靶向这些受体。我们最近的工作是检查 MGluR2/4杂二聚体提供了一个可能的解释：靶向时高效的配体 当同一受体以异二聚体的形式表达时，同二聚体受体通常是无效的 另一杯mGluR。更复杂的是，这些药理反应的变化观察到 MGluR2/4杂二聚体并不适用于所有mGluR杂二聚体，甚至不适用于所有含有 杂二聚体。因此，为了了解mGluR配体如何在大脑中发挥作用，我们必须研究 每对可能的异源二聚体在分离中的药理反应。但这很复杂，因为 每一个mGluR也可以形成同源二聚体，所以任何一对表达的mGluR都有未知的倾向 同-和异二聚化。为了解决这一问题，我们设计了一种新型的二聚体合成控制系统 使用ER保留序列与正交的、分裂的内含子、自切蛋白配对的组合 序列，这将允许表达已知的接近野生型mGluR二聚体的纯群体 组成。我们计划制作一份全面的配体与mGluR二聚体图谱，不仅用于帮助 对实验数据的解释，也是为了改进针对mGluRs的治疗策略 病理学。为实现这些目标，我们将追求以下具体目标：1、建设和发展 使用分裂内含子-内质网保留策略来表征标记的mGluR的完整补充，2到 采用适应的CODA-RET方法来获得平行的异源二聚体特异性G蛋白招募数据， 和3，系统地评估每个可能的mGluR二聚体对 选择性激动剂、竞争性拮抗剂、PAM和NAMS。

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. We plan to generate a comprehensive ligand vs. mGluR dimer atlas to be used to not only aid in interpretation of experimental data but also to improve therapeutic strategies targeting mGluRs for a range of pathologies. To accomplish these goals, we will pursue the following Specific Aims: 1, T To build and characterize the full complement of tagged mGluRs using the split intein-ER retention strategy, 2, To employ an adapted CODA-RET approach to obtain parallel heterodimer specific G protein recruitment data, and 3, To systematically assess the pharmacological responses of each probable mGluR dimer pair to selective agonists, competitive antagonists, PAMs and NAMs.