New Tools for Studying Receptor Dimers

研究受体二聚体的新工具

• 批准号: 10579320
• 负责人: Paul J. Kammermeier
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10579320
• 关键词: Affinity; Agonist; Amino Acid Sequence; Behavior; Biological; Brain; Brain region; Cell membrane; Cicatrix; Clinic; Cloning; Color; Communities; Complex; Data; Dimerization; Drug Screening; Endoplasmic Reticulum; Evaluation; Excision; Exhibits; Exteins; Family; Fluorescence Resonance Energy Transfer; Future; G-Protein-Coupled Receptors; Glutamates; Heterodimerization; Homo; Homodimerization; Knowledge; Libraries; Ligands; Link; Maps; Membrane; Membrane Proteins; Metabotropic Glutamate Receptors; Methods; Neurotransmitters; Oranges; Pathology; Performance; Pharmacology; Physiology; Population; Proteins; RNA Splicing; Receptor Signaling; Research; Shapes; Signal Transduction; Sirolimus; Site; Synaptic Transmission; System; Tail; Testing; Work; design; dimer; druggable target; glycoprotein 41; intein; metabotropic glutamate receptor 2; metabotropic glutamate receptor 4; notch protein; novel; pharmacologic; pre-clinical; receptor; receptor function; response; retention rate; sensor; targeted treatment; tool; trafficking

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.

摘要 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。由于mGluRs在大脑中广泛表达并调节兴奋性， 由于具有可塑性，它们已成为各种病理学的药物靶点的候选者。然而迄今为止， 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 具有良好选择性靶向这些受体的丰富的可用配体。我们最近的工作检查mGluR 2/4 异源二聚体提供了一种可能的解释：当靶向同源二聚体时， 当同一受体表达为与另一mGluR的异源二聚体时，受体通常不起作用。 更复杂的是，在mGluR 2/4异二聚体中观察到的药理学反应的这些变化 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。从而 为了了解任何mGluR配体如何在大脑中发挥作用，我们必须研究其药理学反应， 每个可能的异源二聚体对。但这很复杂，因为每一个mGluR也可以形成 因此，任何一对表达的mGluR将具有未知的同源二聚体和异源二聚体的倾向。 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， 保留序列与正交的、分裂的内含肽、自切除蛋白序列配对，这将允许 已知组成的几乎野生型mGluR二聚体的纯群体的表达。该系统将 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 更准确地描述mGluR靶向配体的作用， 药物筛选此外，这种策略为研究其他二聚化膜蛋白提供了模板。 为此，我们将追求以下具体目标：1-确定我们的性能和局限性 新的真核分裂内含肽系统，和2-产生和测试每个mGluR与分裂内含肽的组合， 内含肽系统