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

代谢型谷氨酸受体（mGluR）是C类G蛋白偶联受体，其作为二聚体起作用。 虽然已知mGluR形成同二聚体，但最近的工作表明它们也可以异二聚化， 但不是杂乱的。因为mGluRs在大脑中广泛表达， 和可塑性，它们已经成为各种病理学的药物靶点的候选者。迄今 然而，临床前数据产生的兴奋并没有导致临床中的mGluR靶向治疗， 尽管存在大量具有良好选择性的靶向这些受体的可用配体。我们最近的研究工作 mGluR 2/4异二聚体提供了一种可能的解释：当靶向 当相同受体表达为异二聚体时， 另一种mGluR进一步复杂化的问题是，这些变化的药理学反应中观察到的， mGluR 2/4异二聚体不能推广到所有mGluR异二聚体，或甚至所有含有mGluR 2的异二聚体。 异二聚体。因此，为了了解任何mGluR配体如何在大脑中发挥作用，我们必须检查 每个可能的异源二聚体对的药理学反应。但这很复杂， 每个mGluR也可以形成同源二聚体，因此任何一对表达的mGluR将具有未知的倾向， 均二聚和异二聚。为了解决这个问题，我们设计了一种新型的二聚体组成控制系统 使用ER保留序列与正交、分裂内含肽、自切除蛋白配对的组合 序列，这将允许表达已知的几乎野生型mGluR二聚体的纯群体。 混合物.我们计划生成一个全面的配体与mGluR二聚体图谱，不仅用于帮助 实验数据的解释，而且还改善了针对一系列mGluRs的治疗策略， 病理学为了实现这些目标，我们将追求以下具体目标：1，T建立和 使用断裂内含肽-ER保留策略表征标记的mGluR的完整补体，2， 采用适应的CODA-RET方法获得平行的异二聚体特异性G蛋白募集数据， 和3，为了系统地评估每种可能的mGluR二聚体对的药理学反应， 选择性激动剂、竞争性拮抗剂、PAM和NAM。

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.