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。     

G蛋白偶联受体及其类型的预测

G蛋白偶联受体是非常重要的信号分子受体,其功能失调会导致许多疾病的产生。在前期工作的基础上,作者将序列特征分析与支持向量机技术结合起来,通过分析序列的特征差异,对G蛋白偶联受体分子及其类型进行识别。首次提取了G蛋白偶联受体对应的mRNA序列的绝对密码子使用频率作为特征,这主要因为它既包含了基因密码子使用偏性的信息,也包含了基因所编码蛋白的氨基酸组成信息。结果显示：在G蛋白偶联受体序列及其类型预测的问题中,设计支持向量机分类器时,最好选择使用包含基因序列绝对密码子使用频率和蛋白序列双联氨基酸使用频率两部分信息的组合特征作为特征,同时采用径向基核作为核函数。     

Quantification of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry

G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interactions of receptors within native cell membranes. As a model system, the GPCR-ligand CXCL12α and its receptor CXCR4 are used. Human-derived Ishikawa cells were deposited onto gold coated slides via Langmuir-Schaefer film deposition and interactions between the receptor CXCR4 on these cells and its ligand CXCL12α were detected via total internal reflection ellipsometry (TIRE). This interaction could be inhibited by application of the CXCR4-binding drug AMD3100. Advantages of this approach are that it allows measurement of interactions in a lipid environment without the need for labelling, protein purification or reconstitution of membrane proteins. This technique is potentially applicable to a wide variety of cell types and their membrane receptors, providing a novel method to determine ligand or drug interactions targeting GPCRs and other membrane proteins.     

Computational Prediction of Alanine Scanning and Ligand Binding Energetics in G-Protein Coupled Receptors

Site-directed mutagenesis combined with binding affinity measurements is widely used to probe the nature of ligand interactions with GPCRs. Such experiments, as well as structure-activity relationships for series of ligands, are usually interpreted with computationally derived models of ligand binding modes. However, systematic approaches for accurate calculations of the corresponding binding free energies are still lacking. Here, we report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 receptor and series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones.     

Assessment and Challenges of Ligand Docking into Comparative Models of G-Protein Coupled Receptors

The rapidly increasing number of high-resolution X-ray structures of G-protein coupled receptors (GPCRs) creates a unique opportunity to employ comparative modeling and docking to provide valuable insight into the function and ligand binding determinants of novel receptors, to assist in virtual screening and to design and optimize drug candidates. However, low sequence identity between receptors, conformational flexibility, and chemical diversity of ligands present an enormous challenge to molecular modeling approaches. It is our hypothesis that rapid Monte-Carlo sampling of protein backbone and side-chain conformational space with Rosetta can be leveraged to meet this challenge. This study performs unbiased comparative modeling and docking methodologies using 14 distinct high-resolution GPCRs and proposes knowledge-based filtering methods for improvement of sampling performance and identification of correct ligand-receptor interactions. On average, top ranked receptor models built on template structures over 50% sequence identity are within 2.9 Å of the experimental structure, with an average root mean square deviation (RMSD) of 2.2 Å for the transmembrane region and 5 Å for the second extracellular loop. Furthermore, these models are consistently correlated with low Rosetta energy score. To predict their binding modes, ligand conformers of the 14 ligands co-crystalized with the GPCRs were docked against the top ranked comparative models. In contrast to the comparative models themselves, however, it remains difficult to unambiguously identify correct binding modes by score alone. On average, sampling performance was improved by 10³ fold over random using knowledge-based and energy-based filters. In assessing the applicability of experimental constraints, we found that sampling performance is increased by one order of magnitude for every 10 residues known to contact the ligand. Additionally, in the case of DOR, knowledge of a single specific ligand-protein contact improved sampling efficiency 7 fold. These findings offer specific guidelines which may lead to increased success in determining receptor-ligand complexes.     

Flow-Mediated On-Surface Reconstitution of G-Protein Coupled Receptors for Applications in Surface Plasmon Resonance Biosensors

To facilitate biosensor studies of G-protein coupled receptors (GPCR) and other membrane proteins, reliable methods for preparation of sensor surfaces with high protein density are required. We present here a method for the easy and rapid immobilization and reconstitution of GPCR on carboxylated dextran surfaces modified with long alkyl groups. Following amine coupling of the detergent-solubilized receptor, lipid/detergent-mixed micelles were adhered as they were injected over the immobilized surface, taking advantage of the integrated flow cells. The detergent was eluted in the subsequent buffer flow and the remaining lipid formed a bilayer on the chip surface. With this procedure, rhodopsin was functionally reconstituted in a lipid environment in approximately 1 min. This method can also be used for the easy formation of pure supported lipid bilayers for use in model membrane interaction studies.     

Amphibian Melanophore Technology as a Functional Screen for Antagonists of G-Protein Coupled 7-Transmembrane Receptors

Xenopus laevis melanophores stably expressing 7-transmembrane G-protein-coupled receptors were established and evaluated, either as a primary screening utility for antagonists of the human calcium receptor, or as a screen to assign function to binding inhibitors of human cannabinoid receptors. Stably or transiently expressing melanophores responded selectively to respective effectors of the human calcium, cannabinoid, and neurokinin-1 receptors. Several selective cannabinoid receptor-binding inhibitors of known potency were characterized as agonists or antagonists of the human peripheral cannabinoid (CB(2)) receptor. The results were consistent with changes in cAMP content of hCB(2)-transfected human embryonic kidney (HEK) cells challenged with the same CB(2)-binding antagonists. A stable melanophore cell line expressing the human calcium receptor was used to screen a compound collection directly for functional antagonists, several of which were confirmed as antagonists in secondary screens by stimulating parathyroid hormone (PTH) secretion from bovine parathyroid cells. The percentage of hits in this cell-based screen was reasonably low (1.2%), indicating minimal interference due to toxic effects and validating melanophores as a primary screening modality. Also described is the development of a novel procedure for cryopreservation and reconstitution of cells retaining functional human receptors. ()     

Prostanoid Receptors Involved in Regulation of the Beating Rate of Neonatal Rat Cardiomyocytes

Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F_2α and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD₂ and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP₁ and EP₃ receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP₁ antagonist). Butaprost (a selective prostanoid EP₂ receptor agonist), misoprostol (a prostanoid EP₂ and EP₃ receptor agonist), 11-deoxy-PGE₁ (a prostanoid EP₂, EP₃ and EP₄ receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP₁ receptors are involved in positive regulation of the beating rate. Prostanoid EP₁ receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP₁ and EP₁ receptors (which positively regulate the spontaneous beating rate).     

Lipid Raft-Mediated Regulation of G-Protein Coupled Receptor Signaling by Ligands which Influence Receptor Dimerization: A Computational Study

G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors; they activate heterotrimeric G-proteins in response to ligand stimulation. Although many GPCRs have been shown to form homo- and/or heterodimers on the cell membrane, the purpose of this dimerization is not known. Recent research has shown that receptor dimerization may have a role in organization of receptors on the cell surface. In addition, microdomains on the cell membrane termed lipid rafts have been shown to play a role in GPCR localization. Using a combination of stochastic (Monte Carlo) and deterministic modeling, we propose a novel mechanism for lipid raft partitioning of GPCRs based on reversible dimerization of receptors and then demonstrate that such localization can affect GPCR signaling. Modeling results are consistent with a variety of experimental data indicating that lipid rafts have a role in amplification or attenuation of G-protein signaling. Thus our work suggests a new mechanism by which dimerization-inducing or inhibiting characteristics of ligands can influence GPCR signaling by controlling receptor organization on the cell membrane.     

Regulation of GSK3 isoforms by phosphatases PP1 and PP2A

Dephosphorylation of phospho GSK3 isoforms, from COS-7 cells, was determined in vitro and in cultured cells in the absence or the presence of okadaic acid and lithium. Our results indicate a preferential dephosphorylation of phospho GSK3α by PP2A phosphatase, whereas dephosphorylation of phospho GSK3β mainly takes place by PP1 phosphatase.     

Bile Acid-Induced Arrhythmia Is Mediated by Muscarinic M2 Receptors in Neonatal Rat Cardiomyocytes

Background

Intrahepatic cholestasis of pregnancy (ICP) is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC), which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM). Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart.

Methods and Results

Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M₂ receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M₂ receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters.

Conclusion

We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M₂ receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.     

理解运动行为抑制调控的新视角：乙酰胆碱门控氯离子通道受体

Acetylcholine, the first identified neurotransmitter, plays crucial roles in various brain functions. One well-known case is its involvement as an activating neurotransmitter in the regulation of locomotion. However, its inhibitory regulatory role, particularly in locomotion, remains poorly understood. In a study conducted by Polat et al., the authors investigated the inhibitory role of acetylcholine in locomotion in C. elegans. In this organism, the acetylcholine-gated chloride channel receptor consists of four subunits. The authors thoroughly examined the loss-of-function of each subunit in movement regulation. Interestingly, the mutant worms were still capable of performing various movements such as forward, backward crawling, and turning, suggesting that the overall movement was not significantly affected. However, quantitative behavior analysis revealed subtle yet significant differences in the timing and postures of the movement in these mutants. Furthermore, the authors employed optogenetics to stimulate a specific neuron involved in backward crawling and demonstrated that the loss-of-function of the receptors in individual neurons affects the transitioning between locomotion modes. This work provides evidence for the inhibitory regulatory role of acetylcholine in locomotion. The loss-of-function of acetylcholine-gated chloride channel receptors likely disrupts the balance of neuronal and circuit physiology, thereby affecting the regulation of locomotion. Moreover, this study highlights the powerful role of quantitative behavior analysis in discovering and understanding more sophisticated functions of neural circuits.     

蛋白磷酸酶2A对微管的调控作用研究进展

微管是细胞骨架的主要成分之一,几乎存在于所有真核生物细胞之中,参与细胞众多生理功能。PP2A是真核生物体内存在最广泛的蛋白磷酸酶之一,可以调控大部分细胞生命活动,其中,包括微管所介导的许多生命活动。该文从以下方面介绍了PP2A在微管功能行使中的重要作用,包括PP2A参与微管蛋白翻译后修饰、调控分子马达和微管相关蛋白的活性、维持细胞周期中微管的动态平衡以及PP2A异常与微管类疾病的相关性。     

Regulation of fission yeast morphogenesis by PP2A activator pta2

Cell polarization is key for the function of most eukaryotic cells, and regulates cell shape, migration and tissue architecture. Fission yeast, Schizosaccharomyces pombe cells are cylindrical and polarize cell growth to one or both cell tips dependent on the cell cycle stage. Whereas microtubule cytoskeleton contributes to the positioning of the growth sites by delivering polarity factors to the cell ends, the Cdc42 GTPase polarizes secretion via actin-dependent delivery and tethering of secretory vesicles to plasma membrane. How growth is restricted to cell tips and how re-initiation of tip growth is regulated in the cell cycle remains poorly understood. In this work we investigated the function of protein phosphatase type 2A (PP2A) in S. pombe morphogenesis by deleting the evolutionary conserved PTPA-type regulatory subunit that we named pta2. pta2-deleted cells showed morphological defects and altered growth pattern. Consistent with this, actin patches and active Cdc42 were mislocalized in the pta2 deletion. These defects were additive to the lack of Cdc42-GAP Rga4. pta2Δ cells show upregulated Cdc42 activity and pta2 interacts genetically with polarisome components Tea1, Tea4 and For3 leading to complete loss of cell polarity and rounded morphology. Thus, regulation of polarity by PP2A requires the polarisome and involves Pta2-dependent control of Cdc42 activity.     

Regulation of Cellular Protein Phosphatase-1 (PP1) by Phosphorylation of the CPI-17 Family, C-kinase-activated PP1 Inhibitors

The regulatory circuit controlling cellular protein phosphatase-1 (PP1), an abundant group of Ser/Thr phosphatases, involves phosphorylation of PP1-specific inhibitor proteins. Malfunctions of these inhibitor proteins have been linked to a variety of diseases, including cardiovascular disease and cancer. Upon phosphorylation at Thr³⁸, the 17-kDa PP1 inhibitor protein, CPI-17, selectively inhibits a specific form of PP1, myosin light chain phosphatase, which transduces multiple kinase signals into the phosphorylation of myosin II and other proteins. Here, the mechanisms underlying PP1 inhibition and the kinase/PP1 cross-talk mediated by CPI-17 and its related proteins, PHI, KEPI, and GBPI, are discussed.     

Regulation of AMPA Receptors by Phosphorylation

The AMPA receptors for glutamate are oligomeric structures that mediate fast excitatory responses in the central nervous system. Phosphorylation of AMPA receptors is an important mechanism for short-term modulation of their function, and is thought to play an important role in synaptic plasticity in different brain regions. Recent studies have shown that phosphorylation of AMPA receptors by cAMP-dependent protein kinase (PKA) and Ca2+- and calmodulin-dependent protein kinase II (CaMKII) potentiates their activity, but phosphorylation of the receptor subunits may also affect their interaction with intracellular proteins, and their expression at the plasma membrane. Phosphorylation of AMPA receptor subunits has also been investigated in relation to processes of synaptic plasticity. This review focuses on recent advances in understanding the molecular mechanisms of regulation of AMPA receptors, and their implications in synaptic plasticity.     

协同刺激因子B7家族成员的抑制性受体与负向免疫调节

维持淋巴细胞的正常功能需要正负向协同刺激信号的同时参与。两种信号决定了T、B细胞对抗原特异性刺激的敏感性和应答方式。二者的平衡使机体在避免对自身抗原产生不适当反应的同时,又能对外来抗原显示足够强的应答能力。多年来有关协同信号的研究,对相关分子结构和功能的认识已大大深化,特别是其中的B7分子及其受体家族。该家族的负向调控作用是通过其抑制性受体来实现的。目前已发现3种抑制性受体：细胞毒性T细胞相关分子(CTLA-4)、程序性死亡分子(PD-1)和B、T细胞弱化因子(BTLA)。对其效应机制的研究,将对免疫调节以及自身免疫、肿瘤免疫和移植免疫产生深远影响。     

Lower rate of genomic variation identified in the trans-membrane domain of monoamine sub-class of Human G-Protein Coupled Receptors: The Human GPCR-DB Database

Background  

We have surveyed, compiled and annotated nucleotide variations in 338 human 7-transmembrane receptors (G-protein coupled receptors). In a sample of 32 chromosomes from a Nordic population, we attempted to determine the allele frequencies of 80 non-synonymous SNPs, and found 20 novel polymorphic markers. GPCR receptors of physiological and clinical importance were prioritized for statistical analysis. Natural variation and rare mutation information were merged and presented online in the Human GPCR-DB database .     

Regulation of Endosome Sorting by a Specific PP2A Isoform

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869–5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205–216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.