Elucidating the stoichiometry of GPCR oligomers

项目摘要/摘要 G蛋白偶联受体(GPCRs)以寡聚体的形式组装，决定了它们不同的功能特性。 然而，我们实际上对这些重要的受体是如何组装的，或者血浆中的脂质是如何组装的知之甚少。 膜是大多数GPCRs发挥作用的地方，它调节着它们的结构和功能。是什么决定了 GPCRs对脂类的选择性以及齐聚与脂类/配体结合事件之间的偶联 仍然是该领域的一个关键知识差距；如果得到解决，将极大地促进我们对 这些受体如何参与正常和病理生理过程。已经超过三年了 几十年来，大量的生化方法已被应用于研究gpr结构，表明 这个受体超家族的重要性。然而，因为生化数据在很大程度上构成了间接 关于结构方面的证据，关于GPCRs是作为二聚体还是更大的功能，该领域存在争议， 以及更复杂的低聚物。在这里，我们建议通过调整新的ION来解决这一长期存在的争论 流动质谱(IM-MS)技术，其中非共价相互作用被保留在质量中 光谱仪，以确定GPCR寡聚体的计量和分布。更确切地说，我们将 应用我们首创的新的IM-MS技术来解析和询问个体的脂质和配体结合 膜蛋白复合体的事件。与其他生物物理方法不同，这些方法允许我们 以无与伦比的分辨率确定配体对GPCR单体和低聚物的影响。作为一名模特 GPCR，我们建议对大鼠神经降压素受体1(NTSR1)进行研究，并获得了 使用这种受体与激动剂(神经降压素多肽)结合的基本数据。天然质谱图揭示了 两组低聚复合体，其中一组与预期的一对一复合体匹配。 有趣的是，第二个复合体揭示了一种独特的化学计量比，与先前关于NTSR1的报告不同 因为它是二聚体。我们的长期目标是测试gpcr寡聚体是动态的和 受结合配体和脂质的调节。此R21应用程序的目标是为以下各项奠定基础 通过优化NTSR1的纯化使我们首次能够针对IM-MS进行解析 NTSR1单体和低聚物的单个配体和脂类结合事件。然后我们提议确定 不同去污剂环境对载脂蛋白及其激动剂结合的NTSR1-TO寡聚体状态的影响 为未来研究结合配体如何决定NTSR1和其他GPCRs的组装奠定了基础 和功能。

Project Summary/Abstract G protein-coupled receptors (GPCRs) assemble as oligomers that determine their distinct functional properties. However, we actually know little about how these important receptors assemble, or how lipids in the plasma membrane, where most GPCRs function, modulate their structure and function. What determines the selectivity of GPCRs towards lipids, and the coupling between oligomerization and lipid/ligand binding events remains a key knowledge gap in the field; one that if addressed will significantly advance our understanding of how these receptors participate in both normal and pathophysiological processes. For more than three decades, an arsenal of biochemical approaches has been applied to investigate GPCR structure, indicative of the importance of this superfamily of receptors. However, because biochemical data largely constitutes indirect evidence on structure, there exists a controversy in the field as to whether GPCRs function as dimers or larger, and more complex, oligomers. Here, we propose to address this long-standing debate by adapting new ion mobility mass spectrometry (IM-MS) technology, whereby non-covalent interactions are preserved in the mass spectrometer, to determine the stiochiometry and distribution of GPCR oligomers. More specifically, we will apply novel IM-MS technology we have pioneered to resolve and interrogate individual lipid and ligand binding events to membrane protein complexes. Unlike other biophysical approaches, these methods allow us to determine the effects of ligands on GPCR monomers and oligomers with unparalleled resolution. As a model GPCR, we propose to work on the rat neurotensin receptor type 1 (NTSR1), and have obtained proof-of- principle data using this receptor bound to agonist (neurotensin peptide). The native mass spectrum reveals two populations of oligomeric complexes, with one of them matching the anticipated one to one complex. Interestingly, the second complex revealed a unique stoichiometry, departing from previous reports of NTSR1 as being dimeric. Our long-term goal is to test the hypothesis that GPCR oligomers are dynamic and modulated by bound ligand and lipid. It is the objective of this R21 application to lay the foundation for those studies by optimizing purification of NTSR1 specifically for IM-MS to allow us for the first time to resolve individual ligand and lipid binding events to NTSR1 monomers and oligomers. We then propose to determine the influence of different detergent environments on the oligomeric states of apo and agonist-bound NTSR1 to lay the groundwork for future studies of how bound ligands determine how NTSR1 and other GPCRs assemble and function.