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: 7194125
• 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/7194125
• 关键词: B lymphocyte; G protein; SDS polyacrylamide gel electrophoresis; biological signal transduction; cellular immunity; chemotaxis; confocal scanning microscopy; cyclic AMP; cytokine receptors; dendritic cells; enzyme linked immunosorbent assay; flow cytometry; gene targeting; genetically modified animals; guanosinetriphosphatase activating protein; human tissue; immunoregulation; in situ hybridization; laboratory mouse; molecular /cellular imaging; platelet derived growth factor; polymerase chain reaction; protein structure function; receptor coupling; receptor expression; southern blotting

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.

我们发现了一个称为 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 亚基以及下游信号传导的解离。 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。 RGS1 过度表达的 B 淋巴细胞无法响应趋化因子 CXCL12 进行迁移。相反，从 Rgs1 基因已被基因靶向破坏的小鼠中获得的 Rgs1 -/- B 细胞对 CXCL12 的趋化反应增强，并且在暴露于趋化因子后无法正确脱敏。此外，与野生型小鼠的 B 细胞相比，这些小鼠的 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、生发中心反应过度以及浆细胞运输不当。我们还证明生发中心 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 的构建体，以敲低 RGS1、RGS2、RGS3、RGS10、RGS13、RGS14 或 RGS16 mRNA 表达。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 CXCL12 的反应。在表达低水平 RGS1 的同一 B 细胞系中减少 RGS1 表达，仅轻微增加对 CXCL13 和 CXCL12 的反应。然而，RGS1 和 RGS13 的减少会显着增强对 CXCL12 和 CXCL13 的反应。此外，双敲低细胞在趋化因子暴露后显示出正确极化的能力受损，并且无法正确定向细胞的前缘。为了补充这些研究，我们最近开始检查 Rgs10 或 Rgs13 基因被破坏的小鼠的免疫功能。 虽然 RGS 蛋白减弱异源三聚体 G 蛋白信号传导，但 G α i 亚基的损失预计将显着改变趋化因子受体信号传导。百日咳毒素可灭活所有三个 G α i 亚基，并阻止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量的 G α i2 和 G α i3。令人惊讶的是，缺乏 G α i2 (Gnai2-/-) 但表达 G α i3 的小鼠在趋化因子受体信号传导方面存在重大缺陷。这些小鼠存在胸腺细胞出口缺陷、淋巴结和派尔氏集结发育缺陷。在过继转移实验中，从这些小鼠中分离出的淋巴细胞对趋化因子的反应非常差，并且对脾脏和淋巴结的归巢能力也很差。对这些小鼠过继转移的 T 细胞和 B 细胞的体内成像显示，它们对高内皮微静脉 (HEV) 的粘附性非常差，并且它们在淋巴结内的速度显着降低。这些研究与 Rgs1-/- 小鼠的研究相结合表明，Gnai2 和 Rgs1 之间的比例在淋巴细胞对趋化因子信号传导的反应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，可作为 G α i 和 G α q 的有效 GTP 酶激活蛋白，并减弱由血管紧张素 II、内皮素-1 和 1-磷酸鞘氨醇触发的信号传导。为了证实 RGS5 的生理重要性，我们培育了 RGS5 基因被破坏的小鼠。这些小鼠可以存活，但与对照组相比体重明显偏轻。初步分析表明这些小鼠也患有低血压。另一种 RGS 蛋白 RGS3 经历广泛的 mRNA 剪接。其中一种称为 PDZ-RGS3 的剪接变体被广泛表达。共聚焦和视频延时显微镜的结合显示，过度表达 PDZ-RGS3 GFP 融合蛋白的细胞未能建立功能性中间体。 PDZ-RGS3 GFP 融合蛋白在细胞周期后期定位于中体。此外，我们还发现了一种 shRNA 构建体，可以减少 PDZ-RGS3 的表达，并且其表达会产生相似的表型。此外，我们还发现 PDZ-RGS3 与 Aurora B 激酶（一种已知参与胞质分裂的激酶）发生共免疫沉淀。我们培育出了 Rgs3 等位基因被破坏的小鼠。迄今为止，我们尚未发现任何可行的 Rgs3-/- 小鼠。对中断妊娠获得的胚胎的研究表明，小鼠在妊娠第 10 天左右就会死亡。迄今为止，病理学研究尚未发现胚胎致死的原因。 RGS14 是 RGS 家族的较大成员，包含 RGS、Rap 相互作用和 GoLoco 结构域。众所周知，RGS14 靶向会导致非常早期的胚胎死亡。使用 RGS14 特异性抗体，我们发现 RGS14 与中心体标记物、中心体中的 γ-微管蛋白共定位。进一步的研究表明，RGS14 是一种核质穿梭蛋白。 RGS14 表达的减少会导致微管减少和细胞活力降低。为了补充我们的体外研究，我们已经开始了一个条件基因靶向项目，这将使我们能够研究 RGS14 在成人淋巴细胞中的功能。我们还开始了几项研究，以检查可能参与异三聚体 G 蛋白信号传导但与七个跨膜受体信号传导无关的其他蛋白质的表达。其中包括某些 G α 亚基； RIC-8，G α 亚基的鸟嘌呤核苷酸交换因子； AGS4，一种包含 3 个 GoLoco 结构域的蛋白质，和 G beta5。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良好表达。 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，我们检查了来自鸡蛋溶菌酶 (HEL) 转基因小鼠的 B 细胞 (TgB) 和在 3 维胶原基质中用 HEL (DC-HEL) 脉冲的脾源性 DC。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相遇，最好用“接吻跑和接触”来描述。导致微观和宏观复合体形成的模型。抗原位于 TgB 细胞和 DC-HEL 之间的接触位点。因此，B 细胞与展示其同源抗原的 DC 有效地相互作用，形成类似于 T 细胞和 DC 之间的免疫突触的稳定微环境。我们还测试了许多 B 淋巴细胞趋化性信号分子的特异性抑制剂。这些研究揭示了 PI-3 激酶、P38 激酶和 BTK 激酶在 B 细胞迁移中的潜在作用。用这些激酶的抑制剂处理小鼠 B 细胞或人 B 细胞，可有效抑制 B 细胞趋化性和体内归巢至淋巴结。 PI-3 激酶抑制剂显着减少 B 细胞粘附到高内皮微静脉 (HEV)，而其他两种抑制剂则轻微影响 B 细胞粘附。