G 蛋白偶联受体寡聚体结构研究进展

G蛋白偶联受体(GPCR)是细胞膜上最大的一类受体,其通过构象变化激活下游G蛋白从而介导细胞响应多种来自内源和外界环境中的信号。自GPCR被发现以来,研究者就一直在努力解析GPCR的构象,x射线晶体衍射技术和GPCR蛋白质结晶技术的发展使得越来越多的GPCR单体在静息状态,以及与不同配体甚至G蛋白结合的晶体结构被成功解析。另一方面,FRET和电子显微技术的运用得到了GPCR二聚化和多聚化的多方面证据。本文将结合近年来该领域的进展,对GPCR寡聚体的结构和构象变化予以系统的综述,这些成果为研究GPCR的功能机制及其特异性的靶点药物开发提供了重要的基础。     

G蛋白耦联受体信号转导的偏向性调控及其机制研究进展

G蛋白耦联受体(G protein-coupled receptors,GPCR)被激活后信号可以经G蛋白或β-arrestin向下游传递,并且受体被激活后,由β-arrestin和G蛋白介导的胞内信号传递存在偏向性(signal bias)。这些发现使得GPCR的信号传递体系被重新认识和定义。配体和受体结合位点的细微差异被认为是这一现象产生的关键原因。现有假说认为,不同配体诱导的受体激活态构象也有不同,并由此导致胞内C末端磷酸化位点的不同。磷酸化位点差别最终决定了下游信号传递的走向。偏向性调控现象在GPCR受体家族中并不罕见,其与细胞的许多关键生理功能的精细调控密切相关。利用偏向性调控特点,有可能在减少GPCR靶点相关性副作用的同时,保留其药理学作用,这为GPCR相关的药物开发带来全新思路。     

G蛋白偶联受体的双聚化及其对受体介导信号转导的影响

近年来发现一些G蛋白偶联受体（GPCR）能在细胞膜上形成同源或异源双聚体,并证实受体的双聚化为一些有重要生理功能的GPCR在细胞膜上的表达和信号转导的启动所必需,进一步研究表明,一些GPCR的双聚化不仅可以改变受体与配体结合的特异性和亲和力,而且影响GPCR介导的信号转导的调控,这些结果提示,GPCR之间以及GPCR与其它蛋白在细胞膜上的相互作用是调控GPCR转导信号的一个新途径。     

果蝇长寿基因methuselah的研究进展

果蝇Drosophila 3号染色体上methuselah （mth）基因发生突变后, 成年果蝇的平均寿命会延长约35%, 并且对一系列外界胁迫因素如饥饿、 高温、 百草枯（可产生强氧化性自由基）的耐受性会显著增强。研究表明mth编码的Mth蛋白属于B家族G蛋白偶联受体（G protein-coupled receptor, GPCR）, 其内源性配体是sun基因编码的小分子肽Stunted。现已发现敲除sun基因或者过表达Mth受体的肽类拮抗剂均能延长果蝇的寿命。Mth受体是目前发现的首个与动物衰老调控相关的GPCR, 该受体除了具有GPCR典型的7次跨膜结构外, 还具有其独特的胞外结构域, 该胞外结构域能够与多种配体结合。Mth受体的生理功能主要体现为： 维持生物体内环境稳态和新陈代谢的平衡, 参与调控果蝇的寿命、 应激反应、 雄性种系干细胞数量和感知运动能力等。目前对Mth受体的研究尚处于起步阶段, 其工作机理的解析对于我们揭示GPCR如何参与寿命的调节具有重要意义, 为我们开发延长人类寿命的新药提供了可能。鉴于此, 本文主要对果蝇Mth受体的结构功能、 配体及其寿命调控信号转导通路等方面做了总结, 并对Mth受体寿命调控信号通路的实用研究价值做了一些展望。     

家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

G蛋白偶联受体转激活酪氨酸激酶受体机制

G蛋白偶联受体(G-protien coupled receptors,GPCRs)和酪氨酸激酶受体(receptor tyrosine kinases,RTKs)是体内两类重要的受体家族,介导着绝大多数信号事件。GPCRs能够"绑架"RTKs进行信号转导,即GPCRs能够在没有外加RTKs配体的情况下激活RTKs,这种现象称为转激活。作为转激活的核心过程,GPCR调控RTK磷酸化主要采取RTK配体依赖模式和非RTK配体依赖模式。不同的G蛋白亚型、酪氨酸磷酸激酶、酪氨酸磷酸酶(protein-tyrosine phosphatases,PTPs)以及活性氧自由基(reactiveoxygen species,ROS)均在此过程中具有重要作用。GPCR和RTK还能形成信号复合体(signaling complex)从而实现蛋白质之间的动态相互作用。对转激活的研究为GPCR靶点药物开发提供了新思路。     

锌敏感受体GPR39在疾病发生与治疗中的作用及其机制

锌是调节人类生理活动和许多代谢过程的必需微量元素,Zn²⁺作为第二信使广泛参与细胞增殖和分化、核酸与蛋白质的合成以及其他许多重要的生理活动。细胞外Zn²⁺可与多种细胞表面蛋白质结合,其中最引人注目的是锌受体,即G蛋白偶联受体39(G protein-coupled receptor 39,GPR39),Zn²⁺是目前已知的唯一的GPR39内源性配体。GPR39能在Zn²⁺的影响下激活Ca²⁺信号,导致ERK1/2、AKT等磷酸化,最终激活ERK/MAPK、AKT/PI3K等下游信号转导通路。该文将对GPR39的结构、功能、信号转导通路以及其在疾病发生与治疗中的作用进行综述。     

G蛋白偶联受体的信号通路多样性及药物开发

正G蛋白偶联受体(G protein-coupled receptor,GPCR)是人类基因组编码的最大膜蛋白家族,包含视紫红质样受体、分泌素受体、谷氨酸类受体、黏附类受体和Frizzled/Taste2受体等5个大类共800多个受体成员[1～5]. GPCR受体家族的概念是由杜克大学的Lefkowitz教授和斯坦福大学的Kobilka在1986年首次提出. Lefkowitz与Kobilka等人[6,7]在克隆β肾上腺素受体后,将其序列与视紫红质进行比较,发现二者具有相似的7次跨膜拓扑结构,他们随即提出所有7次跨膜受体都可能具有偶联G蛋白能力的假说.目前认为,GPCR的共同结构特点是具有保守的7次跨膜α螺旋,通过3个胞内环和3个胞外环相连,其N-末端和C-末端     

甜味分子与G蛋白偶联受体Tas1R2/3的相互作用及激活机制

甜味分子与C家族G蛋白偶联受体(G protein-coupled receptor,GPCR)的成员之一甜味受体相互作用,从而激活受体并引起甜味觉的感知。本文简要总结了甜味受体(taste receptor 2 and 3,Tas1R2/3)的结构与功能、甜味分子与受体相互作用并激活受体的机制,并对甜味受体研究领域的发展前景进行了展望。甜味分子与受体相互作用机制的阐明对于理解甜味觉的产生与GPCR的结构与功能具有重要的意义。此外,甜味受体结构与功能的研究可为有针对性地设计新型甜味化合物提供理论基础。     

基于各向异性网络模型研究δ阿片受体的动力学与关键残基

目的 阿片受体是一种G蛋白偶联受体(GPCR),主要通过变构转导胞外区内源性配体结合信号,使其与胞内区效应蛋白偶联来介导镇痛反应。δ阿片受体(DOP)除了与疼痛控制有关外,还与情绪控制有关,是一个很有吸引力的治疗靶点。本文旨在分析DOP的结构动力学和变构效应。方法 首先利用各向异性网络模型(anisotropic network model,ANM)对DOP进行建模,通过慢运动模式和快运动模式残基涨落探索DOP的结构动力学与功能的关系。然后,结合微扰响应扫描(perturbation-response scanning,PRS)对DOP中与变构通信相关的关键残基进行识别。结果 慢运动模式可以很好地识别DOP的结构以及功能性钠离子结合位点,快运动模式可以识别出对蛋白质结构稳定起重要作用的关键残基。残基运动相关性分析发现胞外/胞内的跨膜螺旋与环状区域之间存在正相关性,这些区域相互作用促进DOP与配体的结合。PRS分析中敏感性高和效应性高的关键残基在DOP的变构通信中发挥重要作用。结论 这项工作有助于加强对δ阿片受体变构通讯机制的理解,并为药物设计提供有价值的信息。     

Structural basis for extracellular interactions between calcitonin receptor-like receptor and receptor activity-modifying protein 2 for adrenomedullin-specific binding

The calcitonin receptor-like receptor (CRLR), a class B GPCR, forms a heterodimer with receptor activity-modifying protein 2 (RAMP2), and serves as the adrenomedullin (AM) receptor to control neovascularization, while CRLR and RAMP1 form the calcitonin gene-related peptide (CGRP) receptor. Here, we report the crystal structures of the RAMP2 extracellular domain alone and in the complex with the CRLR extracellular domain. The CRLR-RAMP2 complex exhibits several intermolecular interactions that were not observed in the previously reported CRLR-RAMP1 complex, and thus the shape of the putative ligand-binding pocket of CRLR-RAMP2 is distinct from that of CRLR-RAMP1. The CRLR-RAMP2 interactions were confirmed for the full-length proteins on the cell surface by site-specific photo-crosslinking. Mutagenesis revealed that AM binding requires RAMP2 residues that are not conserved in RAMP1. Therefore, the differences in both the shapes and the key residues of the binding pocket are essential for the ligand specificity.     

Emerging structural biology of lipid G protein‐coupled receptors

The first crystal structure of a G protein‐coupled receptor (GPCR) was that of the bovine rhodopsin, solved in 2000, and is a light receptor within retina rode cells that enables vision by transducing a conformational signal from the light‐induced isomerization of retinal covalently bound to the receptor. More than 7 years after this initial discovery and following more than 20 years of technological developments in GPCR expression, stabilization, and crystallography, the high‐resolution structure of the adrenaline binding β₂‐adrenergic receptor, a ligand diffusible receptor, was discovered. Since then, high‐resolution structures of more than 53 unique GPCRs have been determined leading to a significant improvement in our understanding of the basic mechanisms of ligand‐binding and ligand‐mediated receptor activation that revolutionized the field of structural molecular pharmacology of GPCRs. Recently, several structures of eight unique lipid‐binding receptors, one of the most difficult GPCR families to study, have been reported. This review presents the outstanding structural and pharmacological features that have emerged from these new lipid receptor structures. The impact of these findings goes beyond mechanistic insights, providing evidence of the fundamental role of GPCRs in the physiological integration of the lipid signaling system, and highlighting the importance of sustained research into the structural biology of GPCRs for the development of new therapeutics targeting lipid receptors.     

Ligand Entry and Exit Pathways in the β2-Adrenergic Receptor

The recently determined crystal structure of the human β₂-adrenergic (β₂AR) G-protein-coupled receptor provides an excellent structural basis for exploring β₂AR-ligand binding and dissociation process. Based on this crystal structure, we simulated ligand exit from the β₂AR receptor by applying the random acceleration molecular dynamics (RAMD) simulation method. The simulation results showed that the extracellular opening on the receptor surface was the most frequently observed egress point (referred to as pathway A), and a few other pathways through interhelical clefts were also observed with significantly lower frequencies. In the egress trajectories along pathway A, the D192-K305 salt bridge between the extracellular loop 2 (ECL2) and the apex of the transmembrane helix 7 (TM7) was exclusively broken. The spatial occupancy maps of the ligand computed from the 100 RAMD simulation trajectories indicated that the receptor-ligand interactions that restrained the ligand in the binding pocket were the major resistance encountered by the ligand during exit and no second barrier was notable. We next performed RAMD simulations by using a putative ligand-free conformation of the receptor as input structure. This conformation was obtained in a standard molecular dynamics simulation in the absence of the ligand and it differed from the ligand-bound conformation in a hydrophobic patch bridging ECL2 and TM7 due to the rotation of F193 of ECL2. Results from the RAMD simulations with this putative ligand-free conformation suggest that the cleft formed by the hydrophobic bridge, TM2, TM3, and TM7 on the extracellular surface likely serves as a more specific ligand-entry site and the ECL2-TM7 hydrophobic junction can be partially interrupted upon the entry of ligand that pushes F193 to rotate, resulting in a conformation as observed in the ligand-bound crystal structure. These results may help in the design of β₂AR-targeting drugs with improved efficacy, as well as in understanding the receptor subtype selectivity of ligand binding in the β family of the adrenergic receptors that share almost identical ligand-binding pockets, but show notable amino acid sequence divergence in the putative ligand-entry site, including ECL2 and the extracellular end of TM7.     

Conformational adaptation of nuclear receptor ligand binding domains to agonists: potential for novel approaches to ligand design

Ligands occupy the core of nuclear receptor (NR) ligand binding domains (LBDs) and modulate NR function. X-ray structures of NR LBDs reveal most NR agonists fill the enclosed pocket and promote packing of C-terminal helix 12 (H12), whereas the pockets of unliganded NR LBDs differ. Here, we review evidence that NR pockets rearrange to accommodate different agonists. Some thyroid hormone receptor (TR) ligands with 5′ extensions designed to perturb H12 act as antagonists, but many are agonists. One mode of adaptation is seen in a TR/thyroxine complex; the pocket expands to accommodate a 5′ iodine extension. Crystals of other NR LBDs reveal that the pocket can expand or contract and some agonists do not fill the pocket. A TRβ structure in complex with an isoform selective drug (GC-24) reveals another mode of adaptation; the LBD hydrophobic interior opens to accommodate a bulky 3′ benzyl extension. We suggest that placement of extensions on NR agonists will highlight unexpected areas of flexibility within LBDs that could accommodate extensions; thereby enhancing the selectivity of agonist binding to particular NRs. Finally, agonists that induce similar LBD structures differ in their activities and we discuss strategies to reveal subtle structural differences responsible for these effects.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.     

Elastic network normal mode dynamics reveal the GPCR activation mechanism

G‐protein‐coupled receptors (GPCR) are a family of membrane‐embedded metabotropic receptors which translate extracellular ligand binding into an intracellular response. Here, we calculate the motion of several GPCR family members such as the M2 and M3 muscarinic acetylcholine receptors, the A_2A adenosine receptor, the β₂‐adrenergic receptor, and the CXCR4 chemokine receptor using elastic network normal modes. The normal modes reveal a dilation and a contraction of the GPCR vestibule associated with ligand passage, and activation, respectively. Contraction of the vestibule on the extracellular side is correlated with cavity formation of the G‐protein binding pocket on the intracellular side, which initiates intracellular signaling. Interestingly, the normal modes of rhodopsin do not correlate well with the motion of other GPCR family members. Electrostatic potential calculation of the GPCRs reveal a negatively charged field around the ligand binding site acting as a siphon to draw‐in positively charged ligands on the membrane surface. Altogether, these results expose the GPCR activation mechanism and show how conformational changes on the cell surface side of the receptor are allosterically translated into structural changes on the inside. Proteins 2014; 82:579–586. © 2013 Wiley Periodicals, Inc.     

NMR structure of the thromboxane A2 receptor ligand recognition pocket.

To overcome the difficulty of characterizing the structures of the extracellular loops (eLPs) of G protein-coupled receptors (GPCRs) other than rhodopsin, we have explored a strategy to generate a three-dimensional structural model for a GPCR, the thromboxane A(2) receptor. This three-dimensional structure was completed by the assembly of the NMR structures of the computation-guided constrained peptides that mimicked the extracellular loops and connected to the conserved seven transmembrane domains. The NMR structure-based model reveals the structural features of the eLPs, in which the second extracellular loop (eLP(2)) and the disulfide bond between the first extracellular loop (eLP(1)) and eLP(2) play a major role in forming the ligand recognition pocket. The eLP(2) conformation is dynamic and regulated by the oxidation and reduction of the disulfide bond, which affects ligand docking in the initial recognition. The reduced form of the thromboxane A(2) receptor experienced a decrease in ligand binding activity due to the rearrangement of the eLP(2) conformation. The ligand-bound receptor was, however, resistant to the reduction inactivation because the ligand covered the disulfide bond and stabilized the eLP(2) conformation. This molecular mechanism of ligand recognition is the first that may be applied to other prostanoid receptors and other GPCRs.     

From G Protein-coupled Receptor Structure Resolution to Rational Drug Design

A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design.     

Soluble mimics of a chemokine receptor: chemokine binding by receptor elements juxtaposed on a soluble scaffold

Despite the broad biological importance of G protein-coupled receptors (GPCRs), ligand recognition by GPCRs remains poorly understood. To explore the roles of GPCR extracellular elements in ligand binding and to provide a tractable system for structural analyses of GPCR/ligand interactions, we have developed a soluble protein that mimics ligand recognition by a GPCR. This receptor analog, dubbed CROSS5, consists of the N-terminal and third extracellular loop regions of CC chemokine receptor 3 (CCR3) displayed on the surface of a small soluble protein, the B1 domain of Streptococcal protein G. CROSS5 binds to the CCR3 ligand eotaxin with a dissociation equilibrium constant of 2.9 +/- 0.8 microM and competes with CCR3 for eotaxin binding. Control proteins indicate that juxtaposition of both CCR3 elements is required for optimal binding to eotaxin. Moreover, the affinities of CROSS5 for a series of eotaxin mutants are highly correlated with the apparent affinities of CCR3 for the same mutants, demonstrating that CROSS5 uses many of the same interactions as does the native receptor. The strategy used to develop CROSS5 could be applied to many other GPCRs, with a variety of potential applications.