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蛋白偶联受体结构生物学进展

G蛋白偶联受体(GPCR)是具有7次跨膜螺旋的细胞整合膜蛋白,它们广泛地参与感光、气味、神经传递以及细胞增殖、分化、迁移等各类生理活动的调控.是现代药物研发的重要靶点.然而,GPCR结构生物学研究却受到高质量蛋白制备、稳定性以及结晶方法等方面的限制.近年来,随着新型膜蛋白表达体系、新型去污剂、膜蛋白纯化及结晶技术的发展.使得G蛋白偶联受体结构解析工作取得了可喜的进展,也为进一步解析更多GPCR精细结构及相关药物研发奠定重要基础.     

G蛋白偶联受体计算研究的进展和前瞻

G蛋白偶联受体(G protein-coupled receptor,GPCR)是含有七个跨膜螺旋的一类重要蛋白,是迄今为止发现的最大的多药物靶标受体超蛋白家族。例如,目前上市药物中有超过30%是以GPCR为靶点的。然而,与GPCR重要性形成强烈反差的是科学界对于其结构与功能的了解非常贫乏,主要原因是通过实验手段来获得GPCR的结构与功能信息极其困难。利用生物信息学方法从基因组规模的数据中识别GPCR并预测三维结构是可行途径之一。基于生物信息学的GPCR研究将为新型药物靶标的筛选和药物的开发提供一定的帮助。本文论述了几种较为典型的GPCR计算方法,并基于已有研究提出可能的创新性研究策略来解决GPCR蛋白识别、跨膜区定位、以及结构和功能预测等问题。     

Anti-CRISPR蛋白AcrVA2的结构生物学研究

大多数古生菌及半数细菌都含有成簇有规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)和CRISPR相关(CRISPR-associated,Cas)蛋白质构成的适应性免疫系统,来抵御外界噬菌体的入侵.而噬菌体为了对抗这种免疫系统,也进化出许多抗CRISPR (anti-CRISPR,Acr)的蛋白质,使得CRISPR-Cas系统受到抑制.来自牛眼莫拉氏菌(Moraxella bovoculi)的AcrVA2是目前发现的可抑制V-A型CRISPR-Cas系统效应蛋白Cas12a发挥切割活性的Acr蛋白之一,其作用机理尚不清楚.本文解析了自由状态的AcrVA2和MbCas12a^620-636-AcrVA2复合物的晶体结构,发现AcrVA2蛋白采用了一种新的α-β折叠结构,且只与自由状态的Cas12a结合.此外,AcrVA2与MbCas12a^620-636的结合主要依靠氢键和盐桥的相互作用力,并通过疏水界面得到进一步稳定.这些结果提示,AcrV...     

G蛋白偶联受体的结构与功能

G蛋白偶联受体(Gprotein-coupled receptor,GPCR)是具有7个跨膜螺旋的蛋白质受体,根据其序列的相似性以及与配基的结合情况,共分为5个亚家族,是人体内最大的蛋白质家族,也是重要的药物靶标。二聚体或寡聚体的形成,以及G蛋白偶联受体多元素参与的信号网络传递模式的研究,打破了传统的配基→G蛋白偶联受体→G蛋白→效应器的这种单一的线性信号传递模式,它的结构与功能的研究对于新药的开发、研制以及推动医药领域的发展起着举足轻重的作用。     

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。     

G蛋白偶联受体的结构与功能研究进展

G蛋白偶联受体(GPCR)是一类具有七个跨膜螺旋结构的超家族,参与机体内许多重要的生物学信号转导过程,其功能异常将引起一系列疾病的发生,因而是一类十分重要的药物作用靶标。近几年来,研究人员在GPCR的三维结构测定方面取得了一系列重要的研究进展,这些突破性成就不仅将GPCR家族各亚型及成员的基本结构特征清晰地呈现给人们,也促使人们对它们的功能展开了新一轮广泛而深入的探索。这为基于结构的药物设计,如具有功能选择性的变构调节剂等新型配体的设计,提供了大量重要的信息。现对最近两年在GPCR结构和功能研究方面所取得的重要进展进行综述,并对基于GPCR结构的药物设计及进一步研究进行了展望。     

G蛋白偶联受体的研究进展

G蛋白偶联受体（GPCRs）,是体内最大的蛋白质超家族。GPCRs的基本结构巳清楚,但高分辨率的三维结构还未得到。根据结构的同源性,GPCRs主要分为A、B、C3族。GPCRs配体的多样性决定配体结合域的多样性。受体分子内相互作用力的破坏,质子化,构象变化,与G蛋白的偶联以及受体二聚化参与了GPCRs的活化过程。GPCRs的活化模式有3种;二态模式、多态模式和顺序结合的构象选择模式。     

胃肠黏膜G蛋白偶联受体5阳性细胞研究进展

干细胞和再生医学的研究已成为自然科学中最为引人注目的领,人类已在胚胎干细胞、部分成体干细胞如骨髓造血干细胞方面取得进展,但在其他成体干细胞研究,特别是胃肠黏膜干细胞研究领进展甚少.富含亮氨酸重复序列的 G 蛋白偶联受体5(LGR5)是 G 蛋白偶联受体超家族的成,在胃肠黏膜隐窝基底有少量表达,其阳性细胞可在体内分化为胃肠黏膜所有类型的细胞,被认为是可能的胃肠黏膜成体干细胞.LGR5阳性细胞的研究,对组织工程、消化道疾病发病机制研究、干细胞治疗、肿瘤治疗等方面有重要意义.     

G蛋白偶联受体40研究进展

G蛋白偶联受体40(G protein-coupled receptor 40, GPR40)是一种具有7个跨膜α螺旋结构的G蛋白偶联受体, 主要分布于胰腺细胞和神经系统细胞。它能与机体内游离的中长链脂肪酸结合, 激活细胞内信号通路, 从而调节其生理功能。在胰岛细胞中, GPR40可被游离脂肪酸激活, 促使细胞内钙离子浓度升高, 进而促进胰岛素释放。根据这一机理, 以GPR40为靶点的激动剂类药物相继被开发, 用于糖尿病治疗。GPR40也参与神经发生过程, 但到目前为止其相关机制尚不清楚。文章从基因结构、表达调控、蛋白配体及应用、生理功能等方面详细介绍了GPR40的研究现状, 总结了目前研究中所遇到的问题, 并对未来的研究热点进行展望。     

Distinct clathrin-coated pits sort different G protein-coupled receptor cargo

Upon activation, many G protein-coupled receptors (GPCRs) internalize by clathrin-mediated endocytosis and are subsequently sorted to undergo recycling or lysosomal degradation. Here we observe that sorting can take place much earlier than previously thought, by entry of different GPCRs into distinct populations of clathrin-coated pit (CCP). These distinct populations were revealed by analysis of two purinergic GPCRs, P2Y(1) and P2Y(12), which enter two populations of CCPs in a mutually exclusive manner. The mechanisms underlying early GPCR sorting involve differential kinase-dependent processes because internalization of P2Y(12) is mediated by GPCR kinases (GRKs) and arrestin, whereas P2Y(1) internalization is GRK- and arrestin-independent but requires protein kinase C. Importantly, the beta(2) adrenoceptor which also internalizes in a GRK-dependent manner also traffics exclusively to P2Y(12)-containing CCPs. Our data therefore reveal distinct populations of CCPs that sort GPCR cargo at the plasma membrane using different kinase-dependent mechanisms.     

G protein-coupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions

The medicinal chemistry and pharmacology of the four subtypes of adenosine receptors (ARs) and the eight subtypes of P2Y receptors (P2YRs, activated by a range of purine and pyrimidine mono- and dinucleotides) has recently advanced significantly leading to selective ligands. X-ray crystallographic structures of both agonist- and antagonist-bound forms of the A(2A)AR have provided unprecedented three-dimensional detail concerning molecular recognition in the binding site and the conformational changes in receptor activation. It is apparent that this ubiquitous cell signaling system has implications for understanding and treating many diseases. ATP and other nucleotides are readily released from intracellular sources under conditions of injury and organ stress, such as hypoxia, ischemia, or mechanical stress, and through channels and vesicular release. Adenosine may be generated extracellularly or by cellular release. Therefore, depending on pathophysiological factors, in a given tissue, there is often a tonic activation of one or more of the ARs or P2YRs that can be modulated by exogenous agents for a beneficial effect. Thus, this field has provided fertile ground for pharmaceutical development, leading to clinical trials of selective receptor ligands as imaging agents or for conditions including cardiac arrhythmias, ischemia/reperfusion injury, diabetes, pain, thrombosis, Parkinson's disease, rheumatoid arthritis, psoriasis, dry eye disease, pulmonary diseases such as cystic fibrosis, glaucoma, cancer, chronic hepatitis C, and other diseases.     

Structural biology of G protein-coupled receptors

More than 60% of the current drugs are based on G protein-coupled receptors. Paradoxically, high-resolution structures are not available to facilitate rational drug design. Difficulties in expression, purification, and crystallization of these transmembrane receptors are the reasons for the low success rate. Recent individual and network-based technology development has significantly improved our knowledge of structural biology and might soon bring a major breakthrough in this area.     

Cell-free production of G protein-coupled receptors for functional and structural studies

G-protein coupled receptors (GPCRs) are key elements in signal transduction pathways of eukaryotic cells and they play central roles in many human diseases. So far, most structural and functional approaches have been limited by the immense difficulties in the production of sufficient amounts of protein samples in conventional expression systems based on living cells. We report the high level production of six different GPCRs in an individual cell-free expression system based on Escherichia coli extracts. The open nature of cell-free systems allows the addition of detergents in order to provide an artificial hydrophobic environment for the reaction. This strategy defines a completely new technique for the production of membrane proteins that can directly associate with detergent micelles upon translation. We demonstrate the efficient overproduction of the human melatonin 1B receptor, the human endothelin B receptor, the human and porcine vasopressin type 2 receptors, the human neuropeptide Y4 receptor and the rat corticotropin releasing factor receptor by cell-free expression. In all cases, the long chain polyoxyethylene detergent Brij78 was found to be highly effective for solubilization and milligram amounts of soluble protein could be generated in less than 24 h. Single particle analysis indicated a homogenous distribution of predominantly protein dimers of the cell-free expressed GPCR samples, with dimensions similar to the related rhodopsin. Ligand interaction studies with the endothelin B receptor and a derivative of its peptide ligand ET-1 gave further evidence of a functional folding of the cell-free produced protein.     

Detecting ligand interactions with G protein-coupled receptors in real-time on living cells

G protein-coupled receptors (GPCRs) are a large group of receptors of great biological and clinical relevance. Despite this, the tools for a detailed analysis of ligand–GPCR interactions are limited. The aim of this paper was to demonstrate how ligand binding to GPCRs can be followed in real-time on living cells. This was conducted using two model systems, the radiolabeled porcine peptide YY (pPYY) interacting with transfected human Y2 receptor (hY2R) and the bombesin antagonist RM26 binding to the naturally expressed gastrin-releasing peptide receptor (GRPR). By following the interaction over time, the affinity and kinetic properties such as association and dissociation rate were obtained. Additionally, data were analyzed using the Interaction Map method, which can evaluate a real-time binding curve and present the number of parallel interactions contributing to the curve. It was found that pPYY binds very slowly with an estimated time to equilibrium of approximately 12 h. This may be problematic in standard end-point assays where equilibrium is required. The RM26 binding showed signs of heterogeneity, observed as two parallel interactions with unique kinetic properties. In conclusion, measuring binding in real-time using living cells opens up for a better understanding of ligand interactions with GPCRs.     

Exosome nanovesicles displaying G protein-coupled receptors for drug discovery

Exosomes are naturally occurring nanovesicles that can be tailored to display a broad range of drug targets, including G protein-coupled receptors. Such vesicles provide a new source of complex membrane proteins that are maintained in their native conformation. Given the difficulties to isolate receptors for drug target validation and discovery, receptor presentation on exosome emerges as a promising new tool for drug screening. The potential of this technology is illustrated here with recombinant exosomes presenting the somatostatin receptor 2 as an example. The receptor-containing vesicles were identified as exosomes since they also bear Lactadherin, a hallmark of exosome nanovesicles. The amount of somatostatin receptor 2 on exosomes was similar to the amount of the most abundant known exosome membrane proteins. The receptor was functional and similar in size to the form found on cell surface. Finally, recombinant exosomes were used in several assay formats that exemplify their capacity as a new receptor presentation platform for drug discovery. These include the induction and detection of antibody as well as screening of antibody repertoires without the need to purify membrane proteins.     

Multiplexing G protein-coupled receptors in microarrays: a radioligand-binding assay

Multiplexing of G protein-coupled receptors (GPCRs) in microarrays promises to increase the efficiency, reduce the costs, and improve the quality of high-throughput assays. However, this technology is still nascent and has not yet achieved the status of "high throughput" or laid claim to handling a large set of receptors. In addition, the technology has been demonstrated only when using fluorescent ligands to detect binding, limiting its application to a subset of GPCRs. To expand the impact of multiplexing on this receptor class, we have developed a radiometric approach to the microarray assay. In these studies, we considered two receptors in the alpha-adrenergic receptor family, alpha2A and alpha2C, and the 125I-labeled agonist clonidine. We demonstrate that microarrays of these receptors can be readily detected (signal/noise ratio approximately 160) using a Typhoon 9210 PhosphorImager. In addition, biochemical characterization shows that ligand-binding profiles and selectivity are preserved with the selective antagonists BRL44408 and ARC239. Importantly, these microarrays use approximately 200- to 400-fold less membrane preparation required by conventional assay methods and allow two or more receptors to be assayed in an area equivalent to a standard well of a microtiter plate. The impact of this approach on screening in drug discovery is discussed.     

Genome-wide prediction of G protein-coupled receptors in Verticillium spp

G protein-coupled receptors (GPCRs) are critical factors in regulating morphogenesis, mating, infection and virulence in fungi. In this study, various computational strategies were applied to identify GPCR-like proteins from the genomes of both Verticillium dahliae and Verticillium albo-atrum. The putative GPCRs were distributed over 13 classes, and significantly, three of those represented novel classes of GPCR-like proteins in fungi. The three novel GPCRs had high levels of identity to their counterparts in higher eukaryotes, including Homo sapiens. The numbers of GPCR-like proteins in the two Verticillium spp. were similar to those seen in other filamentous fungi, such as Magnaporthe grisea, Neurospora crassa and Fusarium graminearum. Additionally, the carbon/amino acid receptors were divided into three different subclasses, indicating that differences among the GPCRs existed not only among different classes but also within classes. In conclusion, the identification and classification of GPCRs and their homology to some well-studied fungi will be an important starting point for future research in Verticillium spp.     

Relationship between the G protein signaling and homologous desensitization of G protein-coupled receptors

Signaling and desensitization of G protein-coupled receptor are intimately related, and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, beta2-adrenergic receptor (beta2AR) in S49 lymphoma cells, alpha-factor pheromone receptor (Ste2p) in Saccharomyces cerevisiae LM102 cells, and dopamine D3 receptor (D3R) in HEK-293 cells. Agonist-induced beta-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of beta2AR, which mediates stimulation of adenylyl cyclase, S49 lymphoma cys- cells that lack the alpha subunit of Gs were used. Stimulation of beta2AR in these cells failed to increase intracellular cAMP, but beta-arrestin translocation still occurred, suggesting that feedback from beta2AR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous alpha subunit of Go (Goalpha) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced beta-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of Galphao and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes.