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类黑视素（MO）信号转导的分子基础，MO是最近在光敏视网膜神经节细胞中发现的光受体，它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，以及潜在的各种疾病，包括睡眠障碍、季节性影响障碍和青光眼，美国国立卫生研究院强调黑视素是未来研究的重点。值得注意的是，黑视蛋白的特性非常类似于无脊椎动物的视紫红质，而不是被广泛研究的脊椎动物的“视觉”视紫红质。相似之处包括密切的一级序列同源性和通过gq蛋白（磷脂酶ec /肌醇三磷酸）途径而不是gt蛋白环核苷酸途径传递信号。重要的是，黑视素及其类似的无脊椎动物视紫红质（如鱿鱼视紫红质（sRh））可作为研究人类细胞中数百种gpcr信号转导机制的模型。这类gpcr通过gq蛋白途径发出信号，并被2-arrestin2而不是更特殊的视觉2-arrestin2抑制。突出的例子包括血清素、组胺、肾上腺素、毒蕈碱和降钙素受体，它们是当前和潜在新药的靶点。黑视蛋白和无脊椎动物视紫红质（而非脊椎动物视紫红质）的一个关键特征是它们的光学双稳定性。这种特性允许它们使用两种不同颜色的光在两种不同的稳定状态之间“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及其与2- arrestin2和gq蛋白形成的复合物在光激活时发生的详细结构变化。这项研究将通过几个先进的FTIR差分技术的应用，其中许多是在我们的实验室开发的，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，包括脊椎动物视紫红质光激活过程中发生的构象变化的第一次详细表征和细菌视紫红质的质子泵送机制。我们最近证明了这种方法也能够检测和表征位于膜蛋白信号受体（如感觉视紫红质II及其同源换能器）之间的界面接触区域的关键残基和内部水分子的结构变化。在初步研究中，我们测量了鱿鱼视紫红质及其2-阻滞蛋白2配合物的静态和时间分辨FTIR差谱。通过同位素编辑，我们可以分别表征受体和2-arrestin2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)在纳米脂粒（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部J. Navarro博士实验室的密切合作得到促进，他将制备sRho/2-arrestin2晶体并进行平行x射线晶体学研究；奈梅根大学的W. DeGrip博士，他的实验室表达和表征了功能性重组黑视素，LLNL的M. Coleman博士和加州大学戴维斯分校的实验室开发了在nlp中表达gpcr的无细胞技术。

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.