Long-term depression in rat CA1-subicular synapses depends on the G-protein coupled mACh receptors

The subiculum, which is the primary target of CA1 pyramidal neurons and sending efferent fibres to many brain regions, serves as a hippocampal interface in the neural information processes between hippocampal formation and neocortex. Long-term depression (LTD) is extensively studied in the hippocampus, but not at the CA1-subicular synaptic transmission. Using whole-cell EPSC recordings in the brain slices of young rats, we demonstrated that the pairing protocols of low frequency stimulation (LFS) at 3 Hz and postsynaptic depolarization of -50 mVelicited a reliable LTD in the subiculum. The LTD did not cause the changes of the paired-pulse ratio of EPSC. Furthermore, it did not depend on either NMDA receptors or voltage-gated calcium channels (VGCCs). Bath application of the G-protein coupled muscarinic acetylcholine receptors (mAChRs) antagonists, atropine or scopolamine, blocked the LTD, suggesting that mAChRs are involved in the LTD. It was also completely blocked by either the Ca2+ chelator BAPTA or the G-protein inhibitor GDP-beta-S in the intracellular solution. This type of LTD in the subiculum may play a particular role in the neural information processing between the hippocampus and neocortex. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Molecular evolution of CXCR1, a G protein-coupled receptor involved in signal transduction of neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1.

Subcellular localization and characterization of G protein-coupled receptor homolog from lymphocystis disease virus isolated in China

G protein-coupled receptors (GPCRs) constitute a large superfamily involved in various types of signal transduction pathways, and play an important role in coordinating the activation and migration of leukocytes to sites of infection and inflammation. Viral GPCRs, on the other hand, can help the virus to escape from host immune surveillance and contribute to viral pathogenesis. Lymphocystis disease virus isolated in China (LCDV-C) contains a putative homolog of cellular GPCRs, LCDV-C GPCR. In this paper, LCDV-C GPCR was cloned, and the subcellular localization and characterization of GPCR protein were investigated in fish cells. LCDV-C GPCR encoded a 325-amino acid peptide, containing a typical seven-transmembrane domain characteristic of the chemokine receptors and a conserved DRY motif that is usually essential for receptor activation. Transient transfection of GPCR-EGFP in fathead minnow (FHM) cells and epithelioma papulosum cyprini (EPC) cells indicated that LCDV-C GPCR was expressed abundantly in both the cytoplasm and nucleoplasm. Transient overexpression of GPCR in these two cells cannot induce obvious apoptosis. FHM cells stably expressing GPCR showed enhanced cell proliferation and significant anchorage-independent growth. The effects of GPCR protein on external apoptotic stimuli were examined. Few apoptotic bodies were observed in cells expressing GPCR treated with actinomycin D (ActD). Quantitative analysis of apoptotic cells indicated that a considerable decrease in the apoptotic fraction of cells expressing GPCR, compared with. the control cells, was detected after exposure to ActD and cycloheximide. These data suggest that LCDV-C GPCR may inhibit apoptosis as part of its potential mechanism in mediating cellular transformation.

水稻G蛋白偶联受体OSGPCR1功能的研究

水稻是世界上最重要的粮食作物，也是单子叶植物的模式植物，它为全球近一半的人口提供食物，但是低温、高盐、干旱等非生物胁迫，每年都会在全世界范围内造成水稻大面积减产。G蛋白介导的信号途径是传递胞外信号比较保守的作用机制之一。动物细胞对于G蛋白及其受体(GPCRs)的研究已经取得了很大的进展。而植物细胞中对它们的研究刚刚起步。本文从越冬稻低温响应芯片上筛选到一个膜蛋白，它编码一个推测的G蛋白偶联受体(G protein-coupledreceptor, GPCR)，据此我们将其命名为OsGPCR1，并对其进行深入研究。 OsGPCR1的cDNA全长为1407bp，编码468个氨基酸，在蛋白水平上的同源性比较结果显示，该基因与动物中研究的比较多的异源三聚体G蛋白偶联受体(G Protein- Coupled Receptor)同源性达到44%。经过跨膜结构域预测表明OsGPCR1具有9TMs结构，以GFP为标签的亚细胞定位表明OsGPCR1定位在膜上。GTP酶活性测定试验表明，OsGPCR1蛋白能够激活水稻RGA的GTP酶活性，此外，以泛素裂解体系为基础的酵母双杂交实验表明，OsGPCR1能够与RGA相互作用。说明OsGPCR1编码的蛋白是水稻中的一个G蛋白偶联受体。 OsGPCR1的表达受低温、干旱、高盐的诱导，但不受ABA，GA，ACC，IAA的诱导。在『F常生长条件下，OsGPCR1在水稻各器官中均有表达，但强弱有所不同。 在拟南芥和水稻中超表达OsGPCR1都能显著增强转基因植物对干旱、高盐、低温的耐受性。而在水稻中抑制OsGPCR1的表达，转基因水稻呈现出干旱、高盐、低温的敏感性。对转基因拟南芥下游基因的分析表明，超表达OsGPCR1能够在非胁迫条件下激活CBF途径中相关基因的表达。结合OsGPCR1不受ABA诱导的表达模式，我们推测OsGPCR1可能是通过不依赖于ABA这条途径而传递信号的。借助超表达和转反义水稻材料，利用水稻全基因组芯片研究OsGPCR1靶基因的结果表明，不论OsGPCR1基因表达量的降低或上升，都导致大约30%的与转运相关的基因的表达量发生改变。这暗示OsGPCR1可能通过囊泡运输传递胞外信号。此外FM4-64对超表达和转反义水稻幼根细胞染色标记后，OsGPCR1反义抑制水稻细胞内囊泡在细胞两端呈聚集状，即形成“BFA区间”，而超表达OsGPCRI水稻细胞内囊泡呈密集状。这些结果都表明OsGPCR1可能通过调控囊泡运输而将胞外胁迫信号传递进胞内。

Composition and evolution of the V2r vorneronasal receptor gene repertoire in mice and rats

Pheromones are chemicals produced and detected by conspecifics to elicit social/sexual physiological and behavioral responses, and they are perceived primarily by the vomeronasal organ (VNO) in terrestrial vertebrates. Two large superfamilies of G protein-coupled receptors, V1rs and V2rs, have been identified as pheromone receptors in vomeronasal sensory neurons. Based on a computational analysis of the mouse and rat genome sequences, we report the first global draft of the V2r gene repertoire, composed of similar to 200 genes and pseudogenes. Rodent V2rs are subject to rapid gene births/deaths and accelerated amino acid substitutions, likely reflecting the species-specific nature of pheromones. Vertebrate V2rs appear to have originated twice prior to the emergence of the VNO in ancestral tetrapods, explaining seemingly inconsistent observations among different V2rs. The identification of the entire V2r repertoire opens the door to genomic-level studies of the structure, function, and evolution of this diverse group of sensory receptors. (c) 2005 Elsevier Inc. All rights reserved.

Genomic organization and sequence analysis of the vomeronasal receptor V2R genes in mouse genome

Two multigene superfamilies, named V1R and V2R, encoding seven-transmembrane-domain G-protein coupled receptors (GPCRs) have been identified as pheromone receptors in mammals. Three V2R gene families have been described in mouse and rat. Here we screened

Morphine induces Beclin 1-and ATG5-dependent autophagy in human neuroblastoma SH-SY5Y cells and in the rat hippocampus

Chronic exposure to morphine can induce drug addiction and neural injury, but the exact mechanism is not fully understood. Here we show that morphine induces autophagy in neuroblastoma SH-SY5Y cells and in the rat hippocampus. Pharmacological approach shows that this effect appears to be mediated by PTX-sensitive G protein-coupled receptors signaling cascade. Morphine increases Beclin 1 expression and reduces the interaction between Beclin 1 and Bcl-2, thus releasing Beclin 1 for its pro-autophagic activity. Bcl-2 overexpression inhibits morphine-induced autophagy, whereas knockdown of Beclin 1 or knockout of ATG5 prevents morphine-induced autophagy. In addition, chronic treatment with morphine induces cell death, which is increased by autophagy inhibition through Beclin 1 RNAi. Our data are the first to reveal that Beclin 1 and ATG5 play key roles in morphine-induced autophagy, which may contribute to morphine-induced neuronal injury.

Adaptive evolution of MRGX2, a human sensory neuron specific gene involved in nociception

MRGX2, a G-protein-coupled receptor, is specifically expressed in the sensory neurons of the human peripheral nervous system and involved in nociception. Here, we studied DNA polymorphism patterns and evolution of the MRGX2 gene in world-wide human populations and the representative nonhuman primate species. Our results demonstrated that MRGX2 had undergone adaptive changes in the path of human evolution, which were likely caused by Darwinian positive selection. The patterns of DNA sequence polymorphisms in human populations showed an excess of derived substitutions, which against the expectation of neutral evolution, implying that the adaptive evolution of MRGX2 in humans was a relatively recent event. The reconstructed secondary structure of the human MRGX2 revealed that three of the four human-specific amino acid substitutions were located in the extra-cellular domains. Such critical substitutions may alter the interactions between MRGX2 protein and its ligand, thus, potentially led to adaptive changes of the pain-perception-related nervous system during human evolution. (c) 2005 Elsevier B.V. All rights reserved.

苯肾上腺素诱导乳鼠心肌细胞蛋白质表达变化的蛋白质组分析

α1-肾上腺素受体(α1-Adrenergic receptor,α1-AR)是G蛋白偶联受体(G-protein coupled receptor,GPCR),也是内源性儿茶酚胺、去甲肾上腺素和肾上腺素最重要的靶受体之一.α1-AR广泛分布于机体的各种器官、组织和细胞中,并介导多种生理效应,如血管收缩、蛋白质合成及心脏变力变时作用等[1,2].很多研究已经证实,α1-AR及其信号转导通路与许多心血管疾病存在密切关系[3,4].蛋白质组学可提供一种发现在疾病情况下异常表达蛋白质的方法,为疾病的早期诊断和愈后判断提供指南,并为针对性疾病治疗提供科学依据.本研究以乳鼠心肌细胞为实验模型,利用双向凝胶电泳和飞行时间质谱分析苯肾上腺素诱导乳鼠心肌细胞表达变化的蛋白质.1实验部分1.1试剂苯肾上腺素(Phenylephine,PE)购自Sigma公司;胰蛋白酶和DMEM购自Hyclone公司;IPG预制胶条(pH=5~8,胶条长17 cm),载体两性电解质(B io-Lyte5-8)购于B io-Rad公司;TPCK修饰的测序级胰酶购自Promega公司;其它试剂均为国产分析纯.1.2实验过程(1)乳鼠心肌细胞培养及样...

Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates

Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemosensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantly with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolutionary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among primates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.

Stress and long-term synaptic depression

Acute inescapable stress reverses the direction of synaptic plasticity in the intact hippocampus via a corticosterone-mediated activation of glucocorticoid receptors and protein/RNA synthesis.