Control Of G Protein Signaling: Role Of The RGSs
G 蛋白信号传导的控制:RGS 的作用
基本信息
- 批准号:7194125
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:B lymphocyteG proteinSDS polyacrylamide gel electrophoresisbiological signal transductioncellular immunitychemotaxisconfocal scanning microscopycyclic AMPcytokine receptorsdendritic cellsenzyme linked immunosorbent assayflow cytometrygene targetinggenetically modified animalsguanosinetriphosphatase activating proteinhuman tissueimmunoregulationin situ hybridizationlaboratory mousemolecular /cellular imagingplatelet derived growth factorpolymerase chain reactionprotein structure functionreceptor couplingreceptor expressionsouthern blotting
项目摘要
We have discovered a protein family termed Regulators of G-protein Signaling (RGS) that impair signal transduction through pathways that use seven trans- membrane receptors and heterotrimeric G proteins. Such receptors, when activated following the binding of a ligand such as a hormone or chemokine, trigger the G alpha subunit to exchange GTP for GDP; this causes the dissociation of G alpha and G beta-gamma subunits and downstream signaling. RGS proteins bind G alpha subunits and function as GTPase activating proteins (GAPs), thereby deactivating the G alpha subunit and facilitating their re-association with G beta-gamma. We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. RGS1 over-expressing B lymphocytes fail to migrate in response to the chemokine CXCL12. Conversely, Rgs1 -/- B cells obtained from mice in which the Rgs1 gene has been disrupted by gene targeting have an enhanced chemotaxic response to CXCL12 and fail to desensitize properly following exposure to chemokines. Furthermore, B cells from these mice enter into lymph nodes more easily, target better into lymph node follicles, and move more rapidly than do B cells from wild type mice. Likely as a consequence the Rgs1 -/- mice have impaired immune responses, altered lymphoid tissue architecture, an excessive germinal center response, and improper trafficking of plasma cells. We have also demonstrated that germinal center B lymphocytes and thymic epithelial cells strongly express another RGS protein, RGS13. To study the role of RGS13 as well as other RGS proteins we have developed constructs that express shRNAs to knock-down RGS1, RGS2, RGS3, RGS10, RGS13, RGS14, or RGS16 mRNA expression. Introduction of a shRGS13 construct into human B cell lines reduces RGS13 mRNA expression and enhances responses to CXCL13 and to CXCL12. Reduction of RGS1 expression in the same B cell line that expresses low levels of RGS1, only mildly increases responses to CXCL13 and CXCL12. However, reduction of both RGS1 and RGS13 dramatically augments the responses to CXCL12 and CXCL13. In addition, the double knock-down cells show an impaired ability to properly polarize following chemokine exposure and an inability to properly orient the leading edge of the cell. To complement these studies we have recently begun to examine immune function in mice in which the Rgs10 or the Rgs13 gene has been disrupted.
While RGS proteins attenuate heterotrimeric G-protein signaling the loss of G alpha i subunits would be expected to dramatically alter chemokine receptor signaling. Pertussis toxin, which inactivates all three G alpha i subunits blocks lymphocyte responses to chemokine stimulation. Lymphocytes predominately express nearly equivalent amounts of G alpha i2 and G alpha i3. Surprisingly mice, which lack G alpha i2 (Gnai2-/-) yet express G alpha i3 have a major defect in chemokine receptor signaling. These mice have a thymocyte egress defect, and defective lymph node and Peyer?s patch development. Lymphocytes isolated from these mice respond very poorly to chemokines and home poorly to the spleen and lymph nodes in adoptive transfer experiments. In vivo imaging of adoptively transferred T and B cells from these mice revealed very poor adhesion to high endothelial venules (HEVs) and a marked reduction in their velocities within lymph nodes. These studies in conjunction with the studies of the Rgs1-/- mice suggest that the ratio between Gnai2 and Rgs1 plays a crucial role in the responsiveness of lymphocytes to chemokine signaling.
Another RGS protein highly expressed in vascular smooth muscle, RGS5, acts as a potent GTPase activating protein for G alpha i and G alpha q and attenuates signaling triggered by angiotensin II, endothelin-1, and sphingosine-1-phosphate. To confirm the physiologic importance of RGS5, mice in which the RGS5 gene has been disrupted have been developed. These mice are viable, but significantly underweight versus controls. Preliminary analysis suggests that these mice are also hypotensive. Another RGS protein, RGS3 undergoes extensive mRNA splicing. One of the splice variants termed PDZ-RGS3 is widely expressed. A combination of confocal and video time-lapse microscopy revealed that cells overexpressing a PDZ-RGS3 GFP fusion protein failed to establish a functional midbody. The PDZ-RGS3 GFP fusion protein localized at the midbody during the late stages of the cell cycle. Furthermore, we identified an shRNA construct that reduced PDZ-RGS3 expression and its expression results in a similar phenotype. In addition we have found that PDZ-RGS3 co-immunoprecipitates with the Aurora B kinase, a kinase known to be involved in cytokinesis. We have produced mice with a disrupted Rgs3 allele. To date we have not identified any viable Rgs3-/- mice. Studies of embryos obtained from interrupted pregnancies indicate that the mice are dying around day 10 of gestation. To date pathological studies have not discovered the reason for the embryonic lethality. RGS14, a larger member of the RGS family, contains an RGS, Rap-interacting, and GoLoco domain. RGS14 targeting is known to cause very early embryonic lethality. Using RGS14-specific antibodies we found that RGS14 co-localized with a centrosome marker, gamma-tubulin in centrosomes. Further studies revealed that RGS14 is a nuclear-cytoplasmic shuttling protein. Reduction in RGS14 expression results in a decrease in microtubules and decreases in cell viability. To complement our in vitro studies we have begun a conditional gene targeting project, which should allow us to study the function of RGS14 in adult lymphocytes. We also have begun several studies to examine the expression of other proteins potentially involved in heterotrimeric G-protein signaling, but not associated with signaling through seven transmembrane receptors. These include certain G alpha subunits; RIC-8, a guanine nucleotide exchange factor for G alpha subunits; AGS4, a protein that contains 3 GoLoco domains, and G beta5. Initial immunoblotting experiments and/or RNA expression studies has shown that each of these proteins in well expressed in lymphocytes.
To further facilitate our studies of B cell migration we have developed new imaging tools that allow us to study B cell migration and the interaction of B cells and dendritic cells in more detail. As a model of B cell-DC interactions we examined B cells (TgB) from hen egg lysozyme (HEL) transgenic mice and spleen-derived DCs pulsed with HEL (DC-HEL) in 3-dimensional collagen matrices. Analysis of the live-cell dynamics revealed autonomous movements and random encounters between TgB cells and DC-HEL best described by a ?kiss-run and engage? model that led to formation of micro- and macro-complexes. Antigen localized at contact sites between TgB cells and DC-HEL. Thus, B cells productively interact with DCs displaying their cognate antigen to form a stable microenvironment similar to the immune synapse between T cells and DC. We have also tested a number of specific inhibitors of signaling molecules on B-lymphocyte chemotaxis. These studies have revealed potential roles for PI-3 kinase, P38 kinase, and BTK kinase in B cell migration. Treatment of either mouse B cells or human B cells with inhibitors of each of theses kinases potently inhibits B cell chemotaxis and in vivo homing to lymph nodes. The PI-3 kinase inhibitor markedly reduces B cell sticking to high endothelial venules (HEVs) while the other two inhibitors mildly affect B cell adhesion.
我们发现了一种称为G蛋白信号传导(RGS)调节剂的蛋白质家族,该蛋白质通过使用七个跨膜受体和异核三聚体G蛋白的途径损害信号转导。这种受体在配体(例如激素或趋化因子)结合后激活时,触发Gα亚基以将GTP换成GDP;这会导致G alpha和Gβ-gamma亚基和下游信号的解离。 RGS蛋白结合Gα亚基并充当GTPase激活蛋白(GAP),从而停用Gα亚基并促进其与Gβ-伽马的重新联系。我们已经表明,RGS蛋白通过包括趋化因子受体在内的多种G蛋白偶联受体调节信号传导。 RGS1过表达的B淋巴细胞无法响应趋化因子CXCL12迁移。相反,从靶向基因靶向的RGS1基因中断的小鼠中获得的RGS1 - / - B细胞具有增强对CXCL12的趋化反应,并且在暴露于趋化因子后无法正确脱敏。此外,这些小鼠的B细胞更容易进入淋巴结,靶向淋巴结卵泡,并且比野生型小鼠的B细胞更快地移动。因此,RGS1 - / - 小鼠的可能受损,免疫反应受损,淋巴组织结构改变,生发中心的过度反应以及血浆细胞的运输不当。我们还证明了生发中心B淋巴细胞和胸腺上皮细胞强烈表达另一种RGS蛋白RGS13。为了研究RGS13以及其他RGS蛋白的作用,我们开发了将SHRNA表达为敲除RGS1,RGS2,RGS3,RGS10,RGS10,RGS13,RGS14,RGS14或RGS16 mRNA mRNA表达的构建体。将SHRGS13构建体引入人B细胞系可降低RGS13 mRNA表达,并增强对CXCL13和对CXCL12的反应。在表达较低水平RGS1的同一B细胞系中RGS1表达的降低,只会轻微增加对CXCL13和CXCL12的反应。但是,RGS1和RGS13的降低大大增加了对CXCL12和CXCL13的响应。此外,双敲门细胞显示出在趋化因子暴露后正确极化的能力受损,并且无法正确定位细胞的前缘。为了补充这些研究,我们最近开始检查RGS10或RGS13基因被破坏的小鼠中的免疫功能。
虽然RGS蛋白减弱了异源三聚体G蛋白信号,但预计G alpha I亚基的损失将极大地改变趋化因子受体信号传导。百日咳毒素,使所有三个GαI亚基都失活均阻断淋巴细胞对趋化因子刺激的反应。淋巴细胞主要表达几乎等效的G alpha I2和GαI3。令人惊讶的是,缺乏GαI2(GNAI2 - / - )但表达GαI3的小鼠在趋化因子受体信号传导方面具有主要缺陷。这些小鼠具有胸腺细胞出口缺陷,有缺陷的淋巴结和Peyer的斑块发育。从这些小鼠中分离出来的淋巴细胞对趋化因子的反应非常差,并且在收养转移实验中对脾脏和淋巴结的回家不良。从这些小鼠中传递的T和B细胞的体内成像表明,对高内皮静脉(HEV)的粘附非常差,并且其淋巴结中其速度显着降低。这些研究与RGS1 - / - 小鼠的研究结合表明,GNAI2和RGS1之间的比率在淋巴细胞对趋化因子信号传导的反应性中起着至关重要的作用。
另一种在血管平滑肌中高度表达的RGS蛋白RGS5充当G alpha I和GαQ的有效GTPase激活蛋白,并减弱由血管紧张素II,Endophelin-1和鞘氨醇1-磷酸触发的信号传导。为了确认RGS5的生理重要性,已经开发了RGS5基因的小鼠。这些小鼠是可行的,但体重不足与对照相比。初步分析表明,这些小鼠也是降低的。另一种RGS蛋白RGS3经历了广泛的mRNA剪接。称为PDZ-RGS3的剪接变体之一被广泛表达。共聚焦和视频延时显微镜的结合表明,过表达PDZ-RGS3 GFP融合蛋白的细胞未能建立功能性中体。 PDZ-RGS3 GFP融合蛋白在细胞周期的后期定位在中体。此外,我们确定了一种降低PDZ-RGS3表达及其表达的shRNA构建体导致相似的表型。此外,我们发现PDZ-RGS3与Aurora B激酶共免疫沉淀,Aurora B激酶是一种已知参与细胞因子的激酶。我们已经生产了带破坏RGS3等位基因的小鼠。迄今为止,我们尚未确定任何可行的RGS3 - / - 小鼠。从中断怀孕获得的胚胎的研究表明,妊娠10天左右,小鼠死亡。迄今为止,病理研究尚未发现胚胎致死性的原因。 RGS14是RGS家族的较大成员,其中包含RGS,说唱互动和Goloco域。 RGS14靶向已知会引起非常早期的胚胎致死性。使用RGS14特异性抗体,我们发现RGS14与中心体中的中心体标记物共定位在中心体中。进一步的研究表明,RGS14是一种核断变型穿梭蛋白。 RGS14表达的降低会导致微管的降低并降低细胞活力。为了补充我们的体外研究,我们已经开始了有条件的基因靶向项目,这应该使我们能够研究RGS14在成人淋巴细胞中的功能。我们还开始进行了几项研究,以检查可能参与异源三聚体G蛋白信号传导的其他蛋白质的表达,但与通过七个跨膜受体的信号无关。这些包括某些Gα亚基; RIC-8,Gα亚基的鸟嘌呤核苷酸交换因子; AGS4,一种包含3个Goloco结构域和Gβ5的蛋白质。最初的免疫印迹实验和/或RNA表达研究表明,这些蛋白质中的每一种都在淋巴细胞中表达良好。
为了进一步促进我们对B细胞迁移的研究,我们开发了新的成像工具,使我们能够更详细地研究B细胞迁移以及B细胞和树突状细胞的相互作用。作为B细胞-DC相互作用的模型,我们从鸡蛋溶菌酶(HEL)转基因小鼠和脾脏衍生的DC中检查了B细胞(TGB),并在3维胶原蛋白基质中脉冲用HEL(DC-HEL)脉冲。对活细胞动力学的分析揭示了TGB细胞和DC-HEL之间的自主运动和随机遇到的遇到,由?接吻和参与?导致形成微型和宏观复合物的模型。抗原位于TGB细胞和直流螺旋之间的接触位点。因此,B细胞与表现出其同源抗原的DC有效相互作用,形成了与T细胞和DC之间的免疫突触相似的稳定的微环境。我们还测试了B淋巴细胞趋化性上信号分子的许多特定抑制剂。这些研究揭示了B细胞迁移中PI-3激酶,p38激酶和BTK激酶的潜在作用。用每种这些激酶的抑制剂对小鼠B细胞或人B细胞的处理,可有效抑制B细胞趋化性和体内归巢中的淋巴结。 PI-3激酶抑制剂显着降低了粘在高内皮静脉(HEV)的B细胞,而其他两个抑制剂则轻度影响B细胞粘附。
项目成果
期刊论文数量(0)
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JOHN H KEHRL其他文献
JOHN H KEHRL的其他文献
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{{ truncateString('JOHN H KEHRL', 18)}}的其他基金
SIGNAL TRANSDUCTION IN B LYMPHOCYTES: INDENTIFICATION OF KEY SIGNALING MOLECULE
B 淋巴细胞中的信号转导:关键信号分子的鉴定
- 批准号:
6288951 - 财政年份:
- 资助金额:
-- - 项目类别:
Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules
B 淋巴细胞中的信号转导:关键信号分子的鉴定
- 批准号:
8555816 - 财政年份:
- 资助金额:
-- - 项目类别:
Signal Transduction In B Lymphocytes: Identification Of
B 淋巴细胞中的信号转导:鉴定
- 批准号:
7302658 - 财政年份:
- 资助金额:
-- - 项目类别:
Analysis of the Functional Roles of a Novel G-alpha Nucleotide Cycle
新型 G-α 核苷酸循环的功能作用分析
- 批准号:
7732614 - 财政年份:
- 资助金额:
-- - 项目类别:
Analysis of the Functional Roles of a Novel G-alpha Nucl
新型 G-α 核的功能作用分析
- 批准号:
7313461 - 财政年份:
- 资助金额:
-- - 项目类别:
Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules
B 淋巴细胞中的信号转导:关键信号分子的鉴定
- 批准号:
7964374 - 财政年份:
- 资助金额:
-- - 项目类别:
Analysis of the Functional Roles of a Novel G-alpha Nucleotide Cycle
新型 G-α 核苷酸循环的功能作用分析
- 批准号:
8555896 - 财政年份:
- 资助金额:
-- - 项目类别:
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