Melanopsin Signal Transduction Studied by FTIR Spectroscopy

描述（由申请人提供）：本项目的总体目标是研究人黑视素（MO）中信号转导的分子基础，MO是最近发现的感光视网膜神经节细胞中的光受体，是昼夜节律和瞳孔反应控制的基础。由于黑视素参与多种生理功能，包括睡眠，精神警觉性，饮食习惯和激素水平，以及可能参与多种疾病，包括睡眠障碍，季节性影响障碍和青光眼，NIH已将黑视素作为未来研究的优先事项。值得注意的是，黑视素的性质非常类似于无脊椎动物的视紫红质，而不是广泛研究的脊椎动物“视觉”视紫红质。相似之处包括一级序列同源性和通过Gq蛋白（磷脂酶C/三磷酸肌醇）途径而不是GT蛋白环核苷酸途径的信号传导。重要的是，黑视素及其类似物无脊椎动物视紫红质，如鱿鱼视紫红质（sRh）作为研究人类细胞中数百个GPCR的信号转导机制的模型。这种GPCR通过Gq-蛋白质途径发出信号，并被2-arrestin 2而不是更专门的视觉2-arrestin抑制。突出的例子包括血清素、组胺、肾上腺素能、毒蕈碱和降钙素受体，它们是当前和潜在新药的靶点。黑视素和无脊椎动物视紫红质而非脊椎动物视紫红质的关键特征是它们的光学双稳态。这种特性允许它们在两种不同的稳定状态之间使用两种不同颜色的光进行“循环”。在这个项目中，我们将利用这种双光子特性来研究黑视素，sRh以及它们与2-arrestin 2和Gq-蛋白形成的复合物在光激活时发生的详细结构变化。这项研究将促进应用几个先进的FTIR差异技术，许多在我们的实验室开发，结合定点诱变和同位素标记。这种方法的应用已经导致了以前的几个里程碑，包括第一次详细的表征脊椎动物视紫红质光活化过程中发生的构象变化和细菌视紫红质的质子泵机制。我们最近已经证明了这种方法的能力，也检测和表征关键残基和内部水分子的结构变化，位于膜蛋白信号受体，如感觉视紫红质II和它的同源换能器之间的界面接触区域。在初步研究中，我们已经测量了静态和时间分辨红外差光谱的鱿鱼视紫红质及其2-arrestin 2复合物。通过使用同位素编辑，我们可以分别表征受体和2-arrestin 2组分的构象变化。拟议的研究也将受益于我们最近开发的方法：i）测量亚皮秒蛋白质变化; ii）使用时间分辨FTIR显微镜探测微量的膜蛋白，包括单晶; iii）在纳米脂质体（NLP）中快速体外表达膜蛋白。这项工作将通过与加尔维斯顿德克萨斯大学医学分支的J. Navarro博士的实验室的密切合作来促进，他将制备sRho/2-arrestin 2晶体并进行平行的X射线晶体学研究;他的实验室已经表达并鉴定了功能性重组黑视素。LLNL和加州大学戴维斯分校的科尔曼，他们的实验室已经开发出无细胞技术来表达NLP中的GPCR。 公共卫生相关性：本研究的主要目的是探讨人类黑视素（最近发现的视网膜光感受器）控制人体内部生物钟和瞳孔反应的机制。了解黑视素是很重要的，因为它涉及关键的生理过程，包括睡眠，精神警觉，饮食习惯和激素水平以及涉及这些过程的疾病。我们实验室开发的先进红外光谱方法的应用将使我们能够确定黑视素的详细分子反应及其与其他蛋白质形成的复合物，以短至万亿分之一秒的时间尺度发光。

DESCRIPTION (provided by applicant): The overall objective of this project is to investigate the molecular basis of signal transduction in human melanopsin (MO), the recently discovered light-receptor in photosensitive retinal ganglion cells, which underlies the control of circadian rhythms and pupillary response. Because melanopsin is involved in a variety of physiological functions including sleep, mental alertness, eating habits, and hormonal levels, as well potentially involved in a variety of disorders including sleep disorders, seasonal affected disorders and glaucoma, the NIH has highlighted melanopsin as a priority for future research. Remarkably, the properties of melanopsin strongly resemble invertebrate rhodopsin instead of the extensively studied vertebrate "visual" rhodopsins. Similarities include a close primary sequence homology and signaling through the Gq-protein (phospholipaseC/inositol triphosphate) pathway instead of the Gt-protein cyclic nucleotide pathway. Importantly, melanopsin and its analog invertebrate rhodopsins such as squid rhodopsin (sRh) serve as models for investigating the signal transduction mechanism in the hundreds of GPCRs in human cells. Such GPCRs signal through the Gq-protein pathway and are inhibited by 2-arrestin2 instead of the more specialized visual 2-arrestin. Prominent examples include serotonin, histamine, adrenergic, muscarinic and calcitonin receptors which are targets of current and potentially new drugs. A key feature of melanopsin and invertebrate rhodopsins but not vertebrate rhodopsins is their optical bistability. This property allows them to be "cycled" between two different stable states using two different colors of light. In this project, we will exploit this two-photon property in order to investigate the detailed structural changes occurring upon light activation in melanopsin, sRh and the complexes they formed with 2- arrestin2 and Gq-protein. This research will be facilitated by the application of several advanced FTIR difference techniques, many developed in our laboratory, in conjunction with site-directed mutagenesis and isotope labeling. Application of this approach has led previously to several milestones including the first detailed characterization of the conformational changes which occur during vertebrate rhodopsin photoactivation and the proton pumping mechanism of bacteriorhodopsin. We have recently demonstrated the ability of this approach to also detect and characterize structural changes in key residues and internal water molecules that lie in the interfacial contact region between membrane protein signaling receptors such as sensory rhodopsin II and its cognate transducer. HtrII In preliminary studies, we have measured static and time resolve FTIR difference spectra of squid rhodopsin and its 2-arrestin2 complex. By using isotope editing, we can characterize conformational changes separately in the receptor and 2-arrestin2 components. The proposed studies will also benefit from our recent development of methods to: i) measure sub-picosecond protein changes; ii) probe minute quantities of membrane proteins including single crystals using time-resolved FTIR microscopy and iii) rapidly in vitro express membrane proteins in nanolipoparticles (NLPs). This work will be facilitated by close collaborations with the laboratories of Dr. J. Navarro at the University of Texas Medical Branch, Galveston who will prepare sRho/2-arrestin2 crystals and perform parallel x-ray crystallographic studies; Dr. W. DeGrip at the University of Nijmegen whose laboratory has expressed and characterized functional recombinant melanopsin and Dr. M. Coleman at the LLNL and UC Davis whose laboratory has developed cell-free techniques to express GPCRs in NLPs. PUBLIC HEALTH RELEVANCE: The overall objective of this project is to investigate the mechanism by which human melanopsin, the recently discovered light-receptor in the retina, controls the body's internal clock as well as pupillary response. Understanding melanopsin is important because it is involved in key physiological processes including sleep, mental alertness, eating habits, and hormonal levels as well as disorders involving these processes. The application of advanced infrared spectroscopic methods developed in our laboratory will allow us to determine the detailed molecular response of melanopsin and the complexes it forms with other proteins to light on time scales as short as one trillionth of a second.