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.

我们发现了一个称为 G 蛋白信号调节因子 (RGS) 的蛋白质家族，它通过涉及七段跨膜受体和异三聚体 G 蛋白的途径损害信号转导。这些受体在与激素或趋化因子等配体结合后被激活，触发 G α 亚基将 GTP 交换为 GDP；这会导致 G α 和 G β γ 亚基和下游信号的解离。 RGS 蛋白结合 G α 亚基并充当 GTP 酶激活蛋白 (GAP)，从而使 G α 亚基失活并促进其与 G β γ 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。我们重点研究 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。这引发了一系列检查淋巴细胞趋化因子受体信号传导的研究，并评估调节信号传导途径对 B 淋巴细胞以及较小程度的 T 细胞运输的影响。从 Rgs1 已被基因靶向破坏的小鼠中获得的 Rgs1 /- B 细胞对趋化因子 CXCL12 和 CXCL13 的反应增强，并且在暴露于这些趋化因子后无法正确脱敏。与野生型小鼠的 B 细胞相比，Rgs1 -/- 小鼠的 B 细胞更容易浸润淋巴结，更好地靶向淋巴结滤泡，并且移动更快。 G α 亚基 Gi 的激活是淋巴细胞趋化性和 RGS1 靶标所必需的。人类和小鼠具有三种 Gi 同种型，尽管 G alphai2（由 Gnai2 编码）和 Galphai3（由 Gnai3 编码）在淋巴细胞中占主导地位。我们发现 Gnai2 -/- T 和 B 细胞在趋化因子诱导的细胞内钙动员、趋化性和淋巴结归巢方面具有严重缺陷，而 Gnai2 +/- T 和 B 细胞则表现出中等缺陷。活体显微镜检查显示 Gnai2 -/- CD4 T 细胞和 B 细胞运动缓慢，缺乏方向性持久性。为了补充这些研究，我们在 B 细胞系中表达了 Galphai1-Yellow 荧光蛋白 (YFP)，并开始建立 Galphai2-YFP 和 Galphai3-YFP 表达细胞系。这些细胞系将用于检查 G αi 蛋白在趋化性和淋巴细胞极化中的作用。 Toll 样受体 (TLR) 信号传导是人类和小鼠 B 细胞的强大调节因子，已知可调节 RGS 蛋白的表达。我们发现，脂多糖 (LPS) 刺激可与 TLR4 结合，增强小鼠 B 细胞 CXCR5 和 CCR7 的表达，增加 Gai2 与 RGS1 的比例，并增加趋化试验中响应的 B 细胞百分比。当转移到受体小鼠体内时，LPS 激活的 B 细胞优先定位于淋巴结滤泡的中心，在那里形成高度极化的细胞簇。细胞追踪研究提供了 B 细胞-B 细胞相互作用和定向细胞迁移的证据。染料稀释研究显示，LPS 刺激的受体小鼠细胞广泛增殖。阻断淋巴结进入表明 LPS 激活的 B 细胞比未刺激的 B 细胞更慢地离开淋巴结。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 在人类 B 细胞中的作用，我们表达了 shRNA，这些 shRNA 在人类 B 细胞系中单独或一起降低了 RGS13 和 RGS1 的表达。减少伯基特淋巴瘤细胞系中的 RGS13 和较小程度的 RGS1 表达，增强了对两种趋化因子 CXCL12 和 CXCL13 的反应，同时减少两种 mRNA 更显着地增强了反应。在用 CXCL12 初次刺激后，双敲低 (KD) 细胞对 CXCL12 或 CXCL13 重新刺激的反应比对照细胞更好。双 KD 细胞还表现出更大的极化倾向，并在趋化因子暴露后形成多个小片状伪足。为了补充这些研究，我们已经开始对 Rgs13 -/- 小鼠进行功能表征。从免疫的 Rgs13 -/- 小鼠获得的 B 细胞对趋化因子的反应增强，并且 Rgs13 -/- 小鼠产生具有增加的 IgG 亲和力的抗体反应。这些小鼠腹膜中的 B1a B 细胞也减少，但 B1b 细胞增加。为了促进 Rgs13 表达细胞的分离和生发中心 B 细胞的体内成像，我们正在生成将 GFP（绿色荧光蛋白）引入 RGS13 基因座的小鼠。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缺陷小鼠，并开始了基因打靶项目来开发RGS19缺陷小鼠。 RGS10 存在三种不同的亚型，它们的细胞内定位和调节 GPCR 信号传导的活性有所不同。两种同工型在转染细胞核内以及原代淋巴细胞中的强烈表达表明，除了调节 GPCR 信号传导之外，RGS10 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 生成了构建体，发现两者主要定位于细胞质中，并具有一些质膜表达。评估 RGS19 在淋巴细胞趋化因子受体信号传导中潜在作用的信号传导研究正在进行中。除了趋化因子受体外，另一组 GPCR 已成为淋巴细胞运输的重要调节因子。这些受体均与磷脂 1-磷酸鞘氨醇 (S1P) 结合。 S1P 受体在血管内皮细胞水平发挥作用，通过控制进入髓窦来调节淋巴结出口，并直接作用于淋巴细胞以促进淋巴结出口。此外，S1P 受体在脾边缘区 B 细胞的定位中发挥作用。我们已经证明 RGS1 会通过几种不同的 S1P 受体有效损害信号传导。目前的研究旨在了解 RGS1、其他 RGS 蛋白以及不同的 G α i 亚型在内皮细胞和淋巴细胞中调节 S1P 信号传导中的作用。许多负责定向细胞迁移和淋巴细胞极化的 Gi 信号传导效应器仍然未知，并且已知的效应器通常在 B 细胞运输中具有不明确的作用。我们发现磷酸肌醇 3-激酶（渥曼青霉素，WMN）、布鲁顿酪氨酸激酶（LFM-A13）和 Jun 激酶（SP600125）的药理抑制剂均显着损害 CXCL12 诱导的小鼠 B 细胞趋化性和人 B 淋巴瘤的趋化性细胞系。每种抑制剂都会损害转移的 B 细胞向外周淋巴结的归巢。对对照和抑制剂处理的小鼠腹股沟淋巴结高位内皮小静脉中 B 细胞的活体成像显示，牢固粘附的 B 细胞数量不同程度减少，其中 WMN 最为有效。为了补充这些研究，我们开发了一种无需进入淋巴结即可对淋巴结内原代 B 细胞进行成像的方法。我们发现标记的B细胞会进入体外保存的300微米淋巴结切片的淋巴结滤泡中。转移前用百日咳毒素处理细胞可抑制细胞进入滤泡。在转移前用 BTK 抑制剂或 WMN 对细胞或切片进行处理未能改变淋巴结滤泡内 B 细胞的运动性；然而，JNK 抑制剂严重抑制了 B 细胞的运动。 虽然我们的大部分研究都集中在淋巴细胞中的 RGS 蛋白，但我们对 Rgs5 感兴趣，这是一种在血管平滑肌和周细胞中发现高水平的 RGS 蛋白。缺乏 Rgs5 的小鼠身材瘦削、血压低，并且出现心脏肥大。