C 族GPCRs 的半胱氨酸富集区的研究进展及药学意义

C族GPCRs是体内重要的受体,参与众多重要的生理和病理进程,并具有复杂的结构和激活机制。在体内该族受体形成组成性的二聚体并具有七螺旋跨膜结构(heptahelical transmembrane domain,HD)、捕蝇草模块(venus flytrap domain,VFT)和半胱氨酸富集区(cysteine-rich domain,CRD)。本文系统介绍了近年来CRD单体的序列和结构解析,以及参与受体激活过程的机制研究的历程和进展。同时也展望了这些基础研究成果对于开发新的更具有成药性的以C族GPCRs为靶点的变构剂的指导意义。     

C族G蛋白偶联受体激活过程

C族G蛋白偶联受体(G protein coupled receptor,GPCR)具有七螺旋跨膜域(heptahelical transmembranedomain,HD)、捕蝇夹域(venusflytrapdomain,VFT)和半胱氨酸富集域(cysteine-rich domain,CRD)等功能域,并在体内组成性形成二聚体。该文介绍C族G蛋白偶联受体激活进程中各功能域的构象变化,以及由此产生的构象学效应。     

胰岛素原C肽作用机制

Ｃ肽是胰岛素原中连接ＡＢ两条链的连接肽。在胰岛 β细胞分泌颗粒中 ,胰岛素原经蛋白酶裂解 ,形成等摩尔由ＡＢ链组成的胰岛素和Ｃ肽 ,然后分泌并进入血液。Ｃ肽的种族差异很大 ,其中人Ｃ肽含 31个氨基酸。在胰岛素原分子中Ｃ肽对胰岛素原分子的折叠、二硫键的正确配对等分子结构的形成起重要作用 ,而血液中游离Ｃ肽的生理功能却一直不清楚。近来的研究发现 ,给予Ｉ型糖尿病大鼠超生理剂量的人Ｃ肽配伍胰岛素治疗 ,能防止血管、神经机能障碍[1] ,长期 (3个月 )给予胰岛素配伍Ｃ肽 ,可使Ｉ型糖尿病病人减少尿白蛋白排泄率 ,改善肾功能和自主…     

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。     

GPCRs 组成性活性的研究进展及意义

GPCRs是体内最大的蛋白质亚家族,其活性涉及体内绝大部分重要的生理和病理进程。研究表明,GPCRs或其突变体能在无配体结合的情况下自发产生部分甚至完全活性程度的生理活性,称之为组成性活性。据此研究者提出了GPCRs激活过程中存在多个中间激活态的观点,从而丰富了配体受体相互作用的经典模型,并使组成性活性突变体(constitutive active mutant,CAM)成为研究GPCRs的新方法。本文系统介绍了组成性活性的研究历程,以及近年来利用CAM的方法研究GPCRs的结构、激活机制和活性调节的历程和进展,和体内组成性活性突变的成因和与疾病的关系,以及CAM在研究药物作用机理和新药研发中的意义。     

β-Arrestins参与GPCRs信号通路的分子机制

β-抑制蛋白(β arrestins)是一类在β肾上腺素受体激酶（βARK)提纯过程中发现的重要支架蛋白和信号调控因子;G蛋白偶联受体（GPCRs）为7次跨膜受体,在细胞信号转导中发挥关键作用,是很多临床药物的作用靶点. β-抑制蛋白作为衔接蛋白,调控GPCRs相关的信号通路,介导GPCRs的脱敏、内化、循环、复敏等生理过程,影响多种疾病的进程. 本文总结了β-抑制蛋白参与GPCRs信号通路的研究进展,侧重阐明了其中的分子机制,以期为开发新一代调控GPCRs功能活性的相关药物提供理论基础.     

甜味机制的研究进展

甜味的感受细胞是味觉细胞,味觉细胞是个双极细胞,味觉受体是一类G蛋白偶联受体,根据甜味物质性质的不同,通过两种途径－－cAMP途径与IP3和DAG途径进行甜味转导。PKA,PKC,味素和转导素在甜味转导中发挥了不同的功能。     

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。     

蛋白酶激活受体-2在中枢神经系统的双重作用

蛋白酶激活受体-2是一种细胞膜表面受体,属于G蛋白偶联受体家族,广泛分布于全身多种组织器官,激活后参与许多疾病的发生和发展过程,尤其在中枢神经系统中所起的作用受到人们越来越多的关注。     

G蛋白偶联受体激活丝裂原活化蛋白激酶的机理

多种Ｇ蛋白偶联受体的均能激活丝裂原活化蛋白激酶。Ｇｉ蛋白偶联受体主要通过其βγ亚基,依赖Ｒａｓ蛋白途径;在大多数哺乳类细胞中Ｇｓ蛋白偶联受体通过ＰＫＡ途径抑制Ｒａｓ依赖的ＭＡＰＫ活化,但在ＣＯＳ－７细胞,Ｇｓ蛋白偶联受体通过ＰＫＡ途径使表达的ＭＡＰＫ活化;Ｇｑ蛋白偶联受体主要通过ＰＫＣ途径依赖或非依赖于Ｒａｓ使ＭＡＰＫ活化。ＭＡＰＫ信号途径中ＥＧＦ受体,酪氨酸激酶及调节蛋白Ｓｈｃ等联级反应蛋白可能     

The activation mechanism of class-C G-protein coupled receptors

Class-C G-protein coupled receptors (GPCRs) represent a distant group among the large family of GPCRs. This class includes the receptors for the main neurotransmitters, glutamate and gamma-aminobutyric acid (GABA), and the receptors for Ca(2+), some taste and pheromone molecules, as well as some orphan receptors. Like any other GPCRs, class-C receptors possess a heptahelical domain (HD) involved in heterotrimeric G-protein activation, but most of them also have a large extracellular domain (ECD) responsible for agonist recognition and binding. In addition, it is now well accepted that these receptors are dimers, either homo or heterodimers. This complex architecture raises a number of important questions. Here we will discuss our view of how agonist binding within the large ECD triggers the necessary change of conformation, or stabilize a specific conformation, of the heptahelical domain leading to G-protein activation. How ligands acting within the heptahelical domain can change the properties of these complex macromolecules.     

Structural basis of activation of bitter taste receptor T2R1 and comparison with Class A G-protein-coupled receptors (GPCRs)

The human bitter taste receptors (T2Rs) are non-Class A members of the G-protein-coupled receptor (GPCR) superfamily, with very limited structural information. Amino acid sequence analysis reveals that most of the important motifs present in the transmembrane helices (TM1-TM7) of the well studied Class A GPCRs are absent in T2Rs, raising fundamental questions regarding the mechanisms of activation and how T2Rs recognize bitter ligands with diverse chemical structures. In this study, the bitter receptor T2R1 was used to systematically investigate the role of 15 transmembrane amino acids in T2Rs, including 13 highly conserved residues, by amino acid replacements guided by molecular modeling. Functional analysis of the mutants by calcium imaging analysis revealed that replacement of Asn-66(2.65) and the highly conserved Asn-24(1.50) resulted in greater than 90% loss of agonist-induced signaling. Our results show that Asn-24(1.50) plays a crucial role in receptor activation by mediating an hydrogen bond network connecting TM1-TM2-TM7, whereas Asn-66(2.65) is essential for binding to the agonist dextromethorphan. The interhelical hydrogen bond between Asn-24(1.50) and Arg-55(2.54) restrains T2R receptor activity because loss of this bond in I27A and R55A mutants results in hyperactive receptor. The conserved amino acids Leu-197(5.50), Ser-200(5.53), and Leu-201(5.54) form a putative LXXSL motif which performs predominantly a structural role by stabilizing the helical conformation of TM5 at the cytoplasmic end. This study provides for the first time mechanistic insights into the roles of the conserved transmembrane residues in T2Rs and allows comparison of the activation mechanisms of T2Rs with the Class A GPCRs.     

The multifaceted regulation and functions of PKM2 in tumor progression

Tumor cells undergo metabolic rewiring from oxidative phosphorylation towards aerobic glycolysis to maintain the increased anabolic requirements for cell proliferation. It is widely accepted that specific expression of the M2 type pyruvate kinase (PKM2) in tumor cells contributes to this aerobic glycolysis phenotype. To date, researchers have uncovered myriad forms of functional regulation for PKM2, which confers a growth advantage on the tumor cells to enable them to adapt to various microenvironmental signals. Here the richness of our understanding on the modulations and functions of PKM2 in tumor progression is reviewed, and some new insights into the paradoxical expression and functional differences of PKM2 in distinct cancer types are offered.     

Effect of heterodimer partner RXRalpha on PPARgamma activation function-2 helix in solution

The structural mechanism of allosteric communication between retinoid X receptor (RXR) and its heterodimer partners remains controversial. As a first step towards addressing this question, we report a nuclear magnetic resonance (NMR) study on the GW1929-bound peroxisome proliferator-activated receptor gamma (PPARγ) ligand-binding domain (LBD) with and without the 9-cis-retinoic acid (9cRA)-bound RXRα LBD. Sequence-specific ¹³C^α, ¹³C^β, and ¹³CO resonance assignments have been established for over 95% of the 275 residues in the PPARγ LBD monomer. The ¹HN, ¹⁵N, and ¹³CO chemical shift perturbations induced by the RXRα LBD binding are located at not only the heterodimer interface that includes the C-terminal residue Y477 but also residues Y473 and K474 in the activation function-2 (AF-2) helix. This result suggests that 9cRA-bound RXRα can affect the PPARγ AF-2 helix in solution and demonstrates that NMR is a powerful new tool for studying the mechanism of allosteric ligand activation in RXR heterodimers.     

The activation mechanism of Hsp26 does not require dissociation of the oligomer

Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.     

Structural analysis of the mechanism of inhibition and allosteric activation of the kinase domain of HER2 protein

Aberrant signaling of ErbB family members human epidermal growth factor 2 (HER2) and epidermal growth factor receptor (EGFR) is implicated in many human cancers, and HER2 expression is predictive of human disease recurrence and prognosis. Small molecule kinase inhibitors of EGFR and of both HER2 and EGFR have received approval for the treatment of cancer. We present the first high resolution crystal structure of the kinase domain of HER2 in complex with a selective inhibitor to understand protein activation, inhibition, and function at the molecular level. HER2 kinase domain crystallizes as a dimer and suggests evidence for an allosteric mechanism of activation comparable with previously reported activation mechanisms for EGFR and HER4. A unique Gly-rich region in HER2 following the α-helix C is responsible for increased conformational flexibility within the active site and could explain the low intrinsic catalytic activity previously reported for HER2. In addition, we solved the crystal structure of the kinase domain of EGFR in complex with a HER2/EGFR dual inhibitor (TAK-285). Comparison with previously reported inactive and active EGFR kinase domain structures gave insight into the mechanism of HER2 and EGFR inhibition and may help guide the design and development of new cancer drugs with improved potency and selectivity.     

Quantifying conformational changes in GPCRs: glimpse of a common functional mechanism

Background

G-protein-coupled receptors (GPCRs) are important drug targets and a better understanding of their molecular mechanisms would be desirable. The crystallization rate of GPCRs has accelerated in recent years as techniques have become more sophisticated, particularly with respect to Class A GPCRs interacting with G-proteins. These developments have made it possible for a quantitative analysis of GPCR geometrical features and binding-site conformations, including a statistical comparison between Class A GPCRs in active (agonist-bound) and inactive (antagonist-bound) states.

Results

Here we implement algorithms for the analysis of interhelical angles, distances, interactions and binding-site volumes in the transmembrane domains of 25 Class A GPCRs (7 active and 18 inactive). Two interhelical angles change in a statistically significant way between average inactive and active states: TM3-TM6 (by -9°) and TM6-TM7 (by +12°). A third interhelical angle: TM5-TM6 shows a trend, changing by -9°. In the transition from inactive to active states, average van der Waals interactions between TM3 and TM7 significantly increase as the average distance between them decreases by >2 Å. Average H-bonding between TM3 and TM6 decreases but is seemingly compensated by an increase in H-bonding between TM5 and TM6. In five Class A GPCRs, crystallized in both active and inactive states, increased H-bonding of agonists to TM6 and TM7, relative to antagonists, is observed. These protein-agonist interactions likely favour a change in the TM6-TM7 angle, which creates a narrowing in the binding pocket of activated receptors and an average ~200 ų reduction in volume.

Conclusions

In terms of similar conformational changes and agonist binding pattern, Class A GPCRs appear to share a common mechanism of activation, which can be exploited in future drug development.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0567-3) contains supplementary material, which is available to authorized users.     

Discovery of human antibodies against the C5aR target using phage display technology

Phage display technologies have been increasingly utilized for the generation of therapeutic, imaging and purification reagents for a number of biological targets. Using a variety of different approaches, we have developed antibodies with high specificity and affinity for various targets ranging from small peptides to large proteins, soluble or membrane-associated as well as to activated forms of enzymes. We have applied this approach to G-protein coupled receptors (GPCRs), often considered difficult targets for antibody therapeutics and targeting. Here we demonstrate the use of this technology for the identification of human antibodies targeting C5aR, the chemoattractant GPCR receptor for anaphylatoxin C5a. The N-terminal region (residues 1-31) of C5aR, one of the ligand binding sites, was synthesized, biotinylated and used as the target for selection. Three rounds of selection with our proprietary human Fab phage display library were performed. Screening of 768 isolates by phage ELISA identified 374 positive clones. Based on sequence alignment analysis, the positive clones were divided into 22 groups. Representative Fab clones from each group were reformatted into IgGs and tested for binding to C5aR-expressing cells, the differentiated U-937 cells. Flow cytometric analysis demonstrated that nine out of 16 reformatted IgGs bound to cells. Competition with a C5aR monoclonal antibody S5/1 which recognizes the same N-terminal region showed that S5/1 blocked the binding of positive cell binders to the peptide used for selections, indicating that the identified cell binding IgGs were specific to C5aR. These antibody binders represent viable candidates as therapeutic or imaging agents, illustrating that phage display technology provides a rapid means for developing antibodies to a difficult class of targets such as GPCRs.