STRUCTURAL STUDIES OF G PROTEIN-COUPLED RECEPTORS

描述（由申请人提供）：G蛋白偶联受体（gpcr）在生物医学中很重要，因为它们是约50%的市售药物的靶标。人们对这些受体的结构和功能在分子或原子上的细节知之甚少。GPCR唯一的三维x射线晶体学模型是基态紫红质的非活性构象。视紫红质（Rhodopsin）是视网膜中的感光蛋白，作为一种典型的GPCR被广泛研究。整体膜蛋白含有7个跨膜螺旋，为其11-顺式视网膜发色团提供结合位点。光子的吸收会引起发色团构象的变化，然后引起受体三级结构的变化。这导致受体细胞质表面的改变，使其同源的G-蛋白转导蛋白（Gt）结合。这就启动了信号转导过程的进一步步骤。大多数gpcr以配体的形式响应分子信号。特定的gpcr结合特定的配体，导致配体特异性的细胞反应。大多数gpcr的配体结合位点与视紫质中的视网膜袋重合。配体的结合引起的构象变化与紫红质中光子的吸收相同，所有gpcr的信号转导的其余分子机制是相似的。我们对GPCR结构和功能的理解将通过本提案的组成部分增加。首先，通过实验探测在不同洗涤条件下分离的紫红质的四级结构来研究激活gpcr的低聚状态。对这些制剂进行生理功能评价。该项目的第二部分将使用单分子力显微镜探测膜与视紫红质或5 -羟色胺5HT1AR受体之间的相互作用，以了解gpcr的动力学和稳定性。活化视紫红质的晶体学研究是该项目的第三部分。该项目的最后一个组成部分需要进一步努力纯化转导蛋白/视紫红质复合体，以进行生化和结构表征。这些项目都为一类重要的蛋白质提供了结构信息，这类蛋白质提供了广泛的实验和理论挑战。蛋白质家族对人类健康的重要性使得这种努力是值得的。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are important in biomedicine because they are the targets of about 50% of commercially available drugs. Only little is known about the structure and function of these receptors in any molecular or atomic detail. The only three-dimensional X-ray crystallographic model for a GPCR is that of ground-state rhodopsin in an inactive conformation. Rhodopsin, the light-sensing protein in retina, has been extensively studied as a prototypical GPCR. The integral membrane protein contains seven trans- membrane helices that provide a binding site for its 11-cis-retinal chromophore. Absorption of a photon triggers a change in the chromophore's conformation and then in the receptor's tertiary structure. This results in alterations on the receptor's cytoplasmic surface that permit binding of transducin (Gt), its cognate G- protein. This initiates further steps in the signal transduction process. Most GPCRs respond to molecular signals in the form of ligands. Binding of specific ligands by specific GPCRs results in a ligand-specific cellular response. The ligand binding site for most GPCRs coincides with the retinal pocket in rhodopsin. Binding of a ligand causes the same kinds of conformational changes as does absorption of a photon in rhodopsin, and the remaining molecular mechanisms for signal transduction are similar for all GPCRs. Our understanding of GPCR structure and function will be increased by components of this proposal. First, the oligomeric state of activated GPCRS will be addressed by experiments probing the quaternary structure of rhodopsin isolated under varying detergent condtions. Assessment of physiological function will be made for these preparations. The second part of the project will use single-molecule force microscopy to probe the interactions between the membrane and rhodopsin or the serotonin 5HT1AR receptor to understand the dynamics and stabilities of GPCRs. Crystallographic studies of activated rhodopsin make up the third part of the project. The last component of the project calls for further efforts in purifying the transducin/rhodopsin complex for biochemical and structural characterization. These projects all provide structural information for an important class of proteins, a class that provides extensive experimental and theoretical challenges. The importance of the protein family for human health makes this effort worthwhile.