Control Of G Protein Signaling: Role Of The RGSs

G 蛋白信号传导的控制：RGS 的作用

• 批准号: 7302657
• 负责人: JOHN H KEHRL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7302657
• 关键词: Control; Protein; Signaling; Role; RGSs

We have discovered a protein family known as Regulators of G-protein Signaling (RGS) that impair signal transduction through pathways that involve seven-segment 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. We have focused on the role of RGS proteins in modulating signaling through lymphocyte chemokine receptors. This has led to a series of studies examining lymphocyte chemokine receptor signaling and an assessment of the effects of modulating the signaling pathway on B lymphocyte and to lesser extent T cell trafficking. Rgs1 /- B cells obtained from mice in which Rgs1 has been disrupted by gene targeting have an enhanced response to the chemokines CXCL12 and CXCL13, and fail to desensitize properly following exposure to these chemokines. B cells from the Rgs1 -/- mice infiltrate lymph nodes more easily, better target lymph node follicles, and move more rapidly than do B cells from wild type mice. Activation of the G alpha subunit Gi is required for lymphocyte chemotaxis and the target of RGS1. Humans and mice have three Gi isoforms although G alphai2 (encoded by Gnai2) and Galphai3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2 -/- T and B cells have profound defects in chemokine-induced mobilization of intracellular calcium, chemotaxis, and lymph node homing, whereas Gnai2 +/- T and B cells exhibit modest defects. Intravital microscopy revealed sluggish Gnai2 -/- CD4 T cell and B cell motility with a lack of directional persistence. To complement these studies we have expressed Galphai1-Yellow fluorescent protein (YFP) in a B cell line and have begun to establish G alphai2-YFP and G alphai3-YFP expressing cell lines as well. These cell lines will be used to examine the roles of the G alphai proteins in chemotaxis and lymphocyte polarization. Toll-like receptor (TLR) signaling, a powerful regulator of human and mouse B cells, is known to regulate RGS protein expression. We found that lipopolysaccharide (LPS) stimulation, which engages TLR4, enhances mouse B cell expression of CXCR5 and CCR7, increases the ratio of Gai2 to RGS1, and increases the percentage of B cells responding in chemotaxis assays. When transferred into recipient mice the LPS-activated B cells preferentially localize to the center of the lymph node follicle, where they form clusters of highly polarized cells. Cell tracking studies provided evidence of B cell-B cell interactions and directed cell migration. Dye dilution studies revealed extensive proliferation of the LPS stimulated cells in the recipient mice. Blocking lymph node ingress indicates that LPS activated B cells exit lymph nodes more slowly that do non-stimulated B cells. Germinal center B lymphocytes strongly express another RGS protein, RGS13. To study the role of RGS13 as well as RGS1 in human B cells we expressed shRNAs that reduced RGS13 and RGS1 expression individually or together in human B cell lines. Reducing RGS13, and to a lesser extent RGS1 expression in a Burkitt?s lymphoma cell line enhanced responsiveness to two chemokines, CXCL12 and CXCL13, while reducing both mRNAs more dramatically augmented the responses. The double knock-down (KD) cells responded better to re-stimulation with CXCL12 or CXCL13 after a primary stimulation with CXCL12 than did the control cells. The double KD cells also exhibited a greater propensity to polarize and developed multiple small lamellipodia following chemokine exposure. To complement these studies we have begun to functionally characterize Rgs13 -/-mice. B cells obtained from immunized Rgs13 -/- mice have heightened responses to chemokines and the Rgs13 -/- mice generate antibody responses with increased IgG affinity. These mice also have a reduction in B1a B cells in the peritoneum, but an increase in B1b cells. To facilitate the isolation of Rgs13-expressing cells and the in vivo imaging of germinal center B cells we are generating mice with GFP (green fluorescent protein) introduced into the RGS13 locus. Two other RGS proteins, RGS10 and RGS19 are strongly expressed in lymphocytes. We have obtained RGS10 deficient mice and have begun a gene targeting project to develop RGS19 deficient mice. Three different isoforms of RGS10 exist and differ in their intracellular localization and their activities in modulating GPCR signaling. The strong expression of two of the isoforms within the nucleus of transfected cells as well as in primary lymphocytes suggests that RGS10 may have other effects on lymphocyte function in addition to the regulation of GPCR signaling. We also generated constructs with mouse and human RGS19-GFP and found that both predominantly localize in the cytosol with some plasma membrane expression. Signaling studies to assess the potential role of RGS19 in lymphocyte chemokine receptor signaling are in progress. In addition to chemokine receptors, another group of GPCRs have emerged as important regulators of lymphocyte trafficking. These receptors all bind the phospholipid sphingosine 1-phosphate (S1P). The S1P receptors function at the level of vascular endothelial cells to regulate lymph node egress by controlling access to the medullary sinus and directly on lymphocytes to promote lymph node exit. In addition, S1P receptors function in the positioning of B cells in the marginal zone of the spleen. We have shown that RGS1 potently impairs signaling through several different S1P receptors. Current studies are directed at understanding the role of RGS1, other RGS proteins, and the different G alpha i isoforms in regulating S1P signaling in endothelial cells and lymphocytes. Many of the effectors of Gi signaling responsible for directed cell migration and lymphocyte polarization remain unknown and known effectors often have ill defined roles in B cell trafficking. We found that pharmacologic inhibitors of phosphoinositide 3-kinases (wortmannin, WMN), Bruton?s tyrosine kinase (LFM-A13), and Jun kinases (SP600125) all significantly impair CXCL12-induced mouse B cell chemotaxis and that of a human B lymphoma cell line. Each of the inhibitors impaired the homing of transferred B cells to peripheral lymph nodes. Intravital imaging of control and inhibitor treated mouse B cells in the inguinal lymph node high endothelial venules demonstrated varying reductions in the number of firmly adherent B cells with WMN being the most effective. To complement these studies we have developed a methodology to image primary B cells within lymph nodes without a requirement for lymph node entry. We have found that labeled B cells will enter into the lymph node follicles of 300 micron lymph node sections maintained in vitro. Treatment of the cells with pertussis toxin prior to transfer inhibits the entry into the follicle. Treatment of the cells or the section prior to transfer with either the BTK inhibitor or WMN failed to alter the motility of B cells within the lymph node follicle; however, the JNK inhibitor profoundly inhibited B cell motility. While most of our studies have focused on RGS proteins in lymphocytes we have had an interest in Rgs5, an RGS protein found at high levels in vascular smooth muscles and in pericytes. Mice deficient in Rgs5 are lean, hypotensive, and develop cardiac hypertrophy.

我们发现了一种被称为G蛋白信号传导调节剂（RGS）的蛋白质家族，该蛋白质通过涉及七个段的反膜受体和异源三聚体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；这导致G alpha和G beta伽马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G alpha亚基并促进其与G beta Gamma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。我们专注于RGS蛋白在通过淋巴细胞趋化因子受体调节信号传导中的作用。这导致了一系列研究，研究了淋巴细胞趋化因子受体信号传导，并评估了调节信号传导途径对B淋巴细胞和T细胞运输程度较小的影响。从基因靶向的RGS1中断的小鼠获得的RGS1 /-B细胞对趋化因子CXCL12和CXCL13的反应增强，并且暴露于这些趋化因子后无法正确脱敏。来自RGS1 - / - 小鼠的B细胞更容易浸润淋巴结，更好地靶向淋巴结卵泡，并且比野生型小鼠的B细胞更快地移动。 Gα亚基GI的激活是淋巴细胞趋化性和RGS1靶标所需的。人类和小鼠具有三种GI同工型，尽管G alphai2（由GNAI2）和Galphai3（由GNAI3编码）在淋巴样细胞中占主导地位。我们发现GNAI2 - / - T和B细胞在趋化因子诱导的细胞内钙，趋化性和淋巴结归巢中具有深远的缺陷，而GNAI2 +/- T和B细胞表现出适度的缺陷。插入式显微镜显示出缓慢的GNAI2 - / - CD4 T细胞和B细胞运动性，缺乏方向持久性。为了补充这些研究，我们在B细胞系中表达了Galphai1-荧光蛋白（YFP），并开始建立G alphai2-YFP和G alphai3-YFP，也表达细胞系。这些细胞系将用于检查G alphaI蛋白在趋化性和淋巴细胞极化中的作用。已知人和小鼠B细胞的强大调节剂Toll样受体（TLR）信号传导可调节RGS蛋白表达。我们发现，吸收TLR4的脂多糖（LPS）刺激增强了CXCR5和CCR7的小鼠B细胞表达，增加了GAI2与RGS1的比率，并增加了在化学治分析中响应的B细胞的百分比。当转移到受体小鼠中时，LPS激活的B细胞优先定位于淋巴结卵泡的中心，在那里它们形成高度极化细胞的簇。细胞跟踪研究提供了B细胞-B细胞相互作用和定向细胞迁移的证据。染料稀释研究表明，受体小鼠中LPS刺激的细胞的广泛增殖。阻断淋巴结入口表明，LPS激活的B细胞出口淋巴结更慢，可导致非刺激的B细胞。生发中心B淋巴细胞强烈表达另一种RGS蛋白RGS13。为了研究RGS13和RGS1在人B细胞中的作用，我们表达了shRNA，在人B细胞系中分别或一起分别或一起降低RGS13和RGS1表达。降低RGS13，并在较小程度上RGS1在Burkitt的淋巴瘤细胞系中的表达提高了对两种趋化因子CXCL12和CXCL13的反应能力，同时降低了这两个mRNA，更大程度地减少了反应。与对照细胞相比，用CXCL12或CXCL13在初级刺激后用CXCL12或CXCL13重新刺激的双重敲低（KD）细胞对对照细胞的反应更好。双KD细胞还表现出更大的倾向，在趋化因子暴露后偏振和发展了多个小薄片。为了补充这些研究，我们已经开始在功能上表征RGS13 - / - 小鼠。从免疫的RGS13 - / - 小鼠获得的B细胞对趋化因子的反应增强，RGS13 - / - 小鼠会产生IgG亲和力增加的抗体反应。这些小鼠的腹膜中B1a B细胞的降低，但B1b细胞的增加。为了促进表达RGS13的细胞和生发中心B细胞的体内成像，我们正在用引入RGS13基因座的GFP（绿色荧光蛋白）的小鼠生成小鼠。另外两种RGS蛋白RGS10和RGS19在淋巴细胞中强烈表达。我们已经获得了RGS10缺乏的小鼠，并开始了一个基因靶向项目，以开发RGS19缺乏小鼠。 RGS10的三种不同的同工型在细胞内定位及其在调节GPCR信号中的活性有所不同。转染细胞核以及原发性淋巴细胞中两个同工型的强表达表明，除了调节GPCR信号传导外，RGS10还可能对淋巴细胞功能具有其他影响。我们还用小鼠和人RGS19-GFP生成构建体，发现两者都以某些质膜表达的形式将其定位在细胞质中。信号传导研究以评估RGS19在淋巴细胞趋化因子受体信号中的潜在作用。除趋化因子受体外，另一组GPCR已成为淋巴细胞运输的重要调节剂。这些受体都结合了1-磷酸磷脂鞘氨醇（S1P）。 S1P受体在血管内皮细胞水平上发挥作用，可通过控制对髓样窦的访问和直接在淋巴细胞上促进淋巴结出口来调节淋巴结出口。另外，S1P受体在脾脏边缘区域的B细胞定位中起作用。我们已经表明，RGS1通过几种不同的S1P受体有效损害信号。当前的研究旨在了解RGS1，其他RGS蛋白和不同GαI同工型在调节内皮细胞和淋巴细胞中S1P信号传导中的作用。负责定向细胞迁移和淋巴细胞极化的GI信号传导的许多效应子仍然未知，已知的效应子通常在B细胞运输中具有不明明确的作用。我们发现，磷酸肌醇3-激酶的药理学抑制剂（Wortmannin，WMN），Bruton？S酪氨酸激酶（LFM-A13）和JUN激酶（SP600125）（SP600125）都显着损害了CXCL12诱导的小鼠B细胞Bell ostosaxis and a a Human b Lymphoma b lymphlome b lymphlome b lymphlomphoma and lymply lymply lymphly lymphly in。每个抑制剂都会损害转移的B细胞向周围淋巴结的归纳。对照和抑制剂处理的小鼠B细胞在腹股沟淋巴结高内皮静脉中的插入成像表明，牢固粘附的B细胞的数量有所不同，WMN是最有效的。为了补充这些研究，我们开发了一种方法来对淋巴结中的原代B细胞进行成像，而无需淋巴结进入。我们发现，标记的B细胞将进入体外维持的300微米淋巴结切片的淋巴结卵泡。在转移之前，用百日咳毒素对细胞进行处理会抑制进入卵泡的进入。用BTK抑制剂或WMN转移之前的细胞或截面的处理未能改变淋巴结卵泡中B细胞的运动性；但是，JNK抑制剂深刻抑制了B细胞运动。 尽管我们的大多数研究都集中在淋巴细胞中的RGS蛋白上，但我们对RGS5感兴趣，这是一种在血管平滑肌肉和周细胞中发现的RGS蛋白。缺乏RGS5的小鼠是瘦，降压和发展心脏肥大的小鼠。