Control Of G Protein Signaling: Role Of The RGSs

我们发现了一个名为G蛋白信号调节因子（RGS）的蛋白质家族，它通过使用七种跨膜受体和异源三聚体G蛋白的途径损害信号转导。这些受体在与配体（如激素或趋化因子）结合后被激活，触发G α亚基将GTP交换为GDP；这导致G α和G β - γ亚基的分离和下游信号传导。RGS蛋白结合G α亚基并作为GTPase激活蛋白（gap）发挥作用，从而使G α亚基失活并促进其与G β - γ的重新结合。我们已经证明RGS蛋白通过多种g蛋白偶联受体（包括趋化因子受体）调节信号传导。过表达RGS1的B淋巴细胞在趋化因子CXCL12的作用下无法迁移。相反，从Rgs1基因被基因靶向破坏的小鼠中获得的Rgs1 -/- B细胞对CXCL12具有增强的趋化反应，并且在暴露于趋化因子后不能正常脱敏。此外，这些小鼠的B细胞比野生型小鼠的B细胞更容易进入淋巴结，更好地靶向淋巴结滤泡，并且移动速度更快。可能的结果是，Rgs1 -/-小鼠免疫反应受损，淋巴组织结构改变，生发中心反应过度，浆细胞运输不当。我们还证实生发中心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，我们开发了表达shrna的蛋白，这些shrna可以敲低RGS1、RGS2、RGS3、RGS10、RGS13、RGS14、RGS16和RGS20 mRNA的表达。shRGS13构建体导入人B细胞系可降低RGS13 mRNA的表达，增强对CXCL13和CXCL12的应答。为了补充这些研究，我们最近开始检查Rgs13被破坏的小鼠的B细胞功能。树突状细胞（DCs）为研究化学引诱剂受体信号和RGS蛋白在调节化学引诱剂反应中的作用提供了一个有用的模型。未成熟dc表达RGS2、RGS10、RGS18和RGS19。Toll受体信号传导导致RGS1、RGS16和RGS20的表达被诱导，RGS14和RGS18的表达下调。RGS1-GFP或RGS18-GFP在CXCL12刺激的dc中的表达表明，这些RGS蛋白显著损害了这些细胞的迁移能力。从Rgs1-/-小鼠制备的树突状细胞也对趋化因子刺激过敏。为了更好地了解B淋巴细胞迁移的机制，我们建立了B细胞系屈光或超迁移到CXCL12或CXCL13的亚系。对趋化信号折射的细胞系倾向于普遍地对许多趋化刺激折射。趋化因子刺激后折光线的Ca++响应被抑制，而超响应线的Ca++响应增加。通过基因芯片分析，比较亲代、屈光性和超迁移系的基因表达模式，发现在低迁移系中RGS1和RGS13表达水平较高。我们已经测试了一些特定的信号分子抑制剂对b淋巴细胞趋化的影响。这些研究揭示了PI-3激酶、P38激酶、JAK激酶和Rho激酶在B细胞迁移中的潜在作用。另一种在血管平滑肌中高度表达的RGS蛋白RGS5，作为一种有效的GTPase激活蛋白，用于Gi α和Gq α，并减弱由血管紧张素II、内皮素-1和鞘氨醇-1-磷酸触发的信号。为了证实RGS5在生理上的重要性，研究人员开发了RGS5基因被破坏的小鼠。我们刚刚开始分析这些老鼠。我们还鉴定并克隆了小鼠RGS-PX1以及另外两个相关蛋白RGS-PX2和RGS-PX3的cDNA。这三种蛋白具有相似的总体结构，包括一个n端疏水区、一个PX相关区（PXA）、一个RGS结构域、一个PX结构域和2个卷曲结构域。这些蛋白的RGS结构域对Ga亚基不具有GAP活性，除了RGS- px1的RGS结构域对Gs具有较弱的活性。通过脂质覆盖测定，我们确定了与这些蛋白质的PX结构域相互作用的特定磷脂。GFP融合蛋白的产生使我们能够确定它们在细胞内的定位。在裂变酵母和果蝇中发现了一个RGS-PX蛋白同源物，在秀丽隐杆线虫中发现了两个同源物。为了更好地理解RGS-PX蛋白在哺乳动物细胞中的作用，分裂酵母RGS-PX异构体被破坏，单倍体酵母的鉴定正在进行中。此外，一项广泛的酵母2杂交分析显示，RGS-PX2的c端部分与参与细胞内蛋白质运输的几种蛋白质有强烈的相互作用。另一种RGS蛋白RGS3经历了广泛的mRNA剪接。其中一种剪接变体PDZ-RGS3被广泛表达。共聚焦显微镜和视频延时显微镜显示，过表达PDZ-RGS3 GFP融合蛋白的细胞无法建立功能性的中间体。相反，子细胞通过细胞间桥保持连接。PDZ-RGS3 GFP融合蛋白在细胞周期后期定位于中体。此外，我们发现了一种shRNA结构可以降低PDZ-RGS3的表达，其表达导致了类似的表型。我们最近获得了Rgs3等位基因断裂的小鼠。到目前为止，我们还没有发现任何Rgs3-/-小鼠，这表明Rgs3可能是正常发育的必要基因。RGS14是RGS家族中较大的成员，包含RGS、rap - interaction和GoLoco域。使用RGS14特异性抗体，我们发现RGS14以细胞周期依赖的方式与中心体标记物γ -微管蛋白共定位。进一步研究表明RGS14是一种核细胞质穿梭蛋白。延时视频显微镜显示，过表达RGS14的细胞既不能进入有丝分裂，也不能完成有丝分裂。RGS14的长时间过表达导致含有多余中心体的多核细胞的形成以及微核的形成，这是染色体不平等分离的标志。RGS14可能通过异源三聚体g蛋白参与中心体/纺锤体的定位。为了进一步促进我们对B细胞迁移的研究，我们开发了许多新的成像工具，使我们能够更详细地研究B细胞迁移以及B细胞与树突状细胞的相互作用。作为B细胞- dc相互作用的模型，我们检测了来自鸡蛋溶菌酶（HEL）转基因小鼠的B细胞（TgB）和在三维胶原基质中使用HEL脉冲的脾源dc （DC-HEL）。活细胞动力学分析揭示了TgB细胞和DC-HEL之间的自主运动和随机相遇，最好的描述是“接吻-运行和接合”模型，导致微观和宏观复合物的形成。对照B细胞有短暂的相互作用。不能与DC-HEL形成宏观配合物。抗原定位于TgB细胞和DC-HEL之间的接触位点，两种细胞类型的肌动蛋白骨架向接触区重新排列。TgB细胞- dc相互作用在两种细胞类型中触发同步Ca2+瞬态。因此，B细胞有效地与显示同源抗原的DC相互作用，形成一个稳定的微环境，类似于T细胞和DC之间的免疫突触。

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 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 that express shRNAs that knock-down RGS1, RGS2, RGS3, RGS10, RGS13, RGS14, RGS16, and RGS20 mRNA expression. Introduction of a shRGS13 construct into human B cell lines reduces RGS13 mRNA expression and enhances responses to CXCL13 and more dramatically to CXCL12. To complement these studies we have recently begun to examine B cell function in mice in which Rgs13 has been disrupted. Dendritic cells (DCs) provide a useful model for studying chemoattractant receptor signaling and the role of RGS proteins in regulating chemoattractant responses. Immature DCs expressed RGS2, RGS10, RGS18, and RGS19. Toll receptor signaling resulted in the induction of RGS1, RGS16, and RGS20 and the downregulation of RGS14 and RGS18. Expression of RGS1-GFP or RGS18-GFP in DCs stimulated with CXCL12 revealed that these RGS proteins significantly impair the migratory capacity of these cells. Dendritic cells prepared from Rgs1-/- mice are also hypersensitive to chemokine stimulation. In order to better understand the mechanisms underlying B-lymphocyte migration, sublines of a B cell line refractive or hyper-migratory to either CXCL12 or CXCL13 were developed. Cell lines refractive to chemotactic signaling tended to be universally refractive to many chemotactic stimuli. The Ca++ responses following chemokine stimulation in the refractive line were inhibited while an increased response was observed in the hyper-responsive lines. Comparisons of the gene expression patterns, determine by gene chip analysis, between the parental, refractive and hyper-migrational lines revealed high levels of RGS1 and RGS13 in the hypo-migratory. We have 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, JAK kinases, and Rho kinase in B cell migration. Another RGS protein highly expressed in vascular smooth muscle, RGS5, acts as a potent GTPase activating protein for Gi alpha and Gq alpha 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. We have just begun to analyze these mice. We also identified and cloned a cDNA for murine RGS-PX1 as well as two other related proteins termed RGS-PX2 and RGS-PX3. All three proteins possess a similar overall structure with an n-terminal hydrophobic region, a PX-associated region (PXA), an RGS domain, a PX domain, and 2 coiled-coiled domains. The RGS domains of these proteins do not possess GAP activity for Ga subunits, with the exception of weak activity of the RGS domain of RGS-PX1 for Gs. Using lipid overlay assays we identified the specific phospholipids that interact with the PX domains of these proteins. The production of GFP fusion proteins allowed us to determine their intracellular localization. A RGS-PX protein homolog was found in fission yeast and Drosophila and two in C. elegans. To facilitate the understanding of the role of RGS-PX proteins in mammalian cells, the fission yeast RGS-PX isoform was disrupted and characterization of haploid yeast is in progress. In addition an extensive yeast 2-hybrid analysis revealed that the c-terminal portion of RGS-PX2 interacts strongly with several proteins involved in intracellular protein trafficking. 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. Instead, daughter cells remain connected by intercellular bridges. 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 resulted in a similar phenotype. We have recently obtained mice with a disrupted Rgs3 allele. To date we have not identified any Rgs3-/- mice suggesting that Rgs3 may be an essential gene for normal development. RGS14, a larger member of the RGS family, contains an RGS, Rap-interacting, and GoLoco domain. Using RGS14-specific antibodies we found that RGS14 co-localized with a centrosome marker, gamma-tubulin in centrosomes in a cell cycle-dependent manner. Further studies revealed that RGS14 is a nuclear-cytoplasmic shuttling protein. Time-lapse video microscopy showed that cells over-expressing RGS14 failed either to enter mitosis or to complete mitosis. Prolonged over-expression of RGS14 resulted in formation of multinucleated cells containing supernumerary centrosomes as well as formation of micronuclei, a hallmark of unequal chromosome segregation. RGS14 may play a role in proper positioning of centrosomes/spindle via heterotrimeric G-proteins. To further facilitate our studies of B cell migration we have developed a number of 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. Control B cells had short-lived interactions and didn?t form macro-complexes with DC-HEL. Antigen localized at contact sites between TgB cells and DC-HEL and both cell types rearranged their actin cytoskeleton toward the contact zone. The TgB cell-DC interaction triggered synchronous Ca2+-transients in both cell types. 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.