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: 8336110
• 负责人: JOHN H KEHRL
• 金额: $ 67.35万
• 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/8336110
• 关键词: Adult; Affect; Antibody Formation; Antigens; Antiviral Agents; Anxiety Disorders; Architecture; Atypical lymphocyte; B-Cell Development; B-Cell Lymphomas; B-Lymphocytes; Binding; Bone Marrow; CD3 Antigens; Calcium; Celiac Disease; Cell Proliferation; Cell Surface Receptors; Cell physiology; Cells; Centroblast; Chemotaxis; Chimera organism; Code; Colitis; Defect; Dendritic Cells; Disease; Dissociation; Elements; Embryo; Employee Strikes; Event; Exhibits; Flow Cytometry; Functional disorder; G Beta Gamma; G-Protein-Coupled Receptors; GTP Binding; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Gastrointestinal tract structure; Gene Expression; Genes; Goals; Greater sac of peritoneum; Green Fluorescent Proteins; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hematopoietic; Hematopoietic stem cells; Heterotrimeric GTP-Binding Proteins; Homing; Human; Human G(i) Alpha Proteins; Hypertension; IgG3; Image; Immune; Immune system; Immunity; Immunization; Immunoglobulin M; In Vitro; Inflammatory; Inflammatory disease of the intestine; Insulin-Dependent Diabetes Mellitus; Interleukin-2; Investigation; Knock-in Mouse; Ligand Binding; Link; London; Lymphocyte; Lymphoid; Lymphoid Cell; Mammalian Cell; Mature B-Lymphocyte; Mature T-Lymphocyte; Messenger RNA; Michigan; Minor; Modeling; Multiple Sclerosis; Mus; Mutation; Nucleotides; Pathogenesis; Peripheral; Peritoneal Macrophages; Peritoneum; Peritonitis; Phenotype; Play; Production; Property; Protein Family; Protein Isoforms; RGS Proteins; RGS1 gene; RGS19 gene; RGS2 gene; Receptor Signaling; Regulation; Resistance; Resources; Role; Sequence Homology; Serum; Severities; Signal Pathway; Signal Transduction; Signaling Protein; Site; Sorting - Cell Movement; Spleen; Splenomegaly; Staging; Structure of aggregated lymphoid follicle of small intestine; Structure of germinal center of lymph node; T-Cell Leukemia; T-Cell Proliferation; T-Lymphocyte; Thymus Gland; Translating; Universities; Wild Type Mouse; base; cell motility; cell type; chemokine; chemokine receptor; college; crosslink; desensitization; dimer; gain of function; gastrointestinal; in vivo; insight; intraepithelial; intravital microscopy; knock-down; loss of function; lymph nodes; mRNA Expression; mast cell; medical schools; melanoma; mouse model; neutrophil; patch clamp; peripheral blood; progenitor; protein expression; protein function; receptor; reconstitution; research study; response; selective expression; thymocyte; trafficking

We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. Chemokine receptors signal predominantly by triggering G alpha i nucleotide exchange. Humans and mice have three Gi isoforms although Gi alpha2 (encoded by Gnai2) and Gi alpha3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2 -/- T and B cells have severe defects in chemokine-receptor signaling while Gnai2 +/- T and B cells exhibit modest defects. In vivo, the Gnai2-/- B cells fail to properly access lymph node follicles and the Gnai2-/- T cells failing to properly enter the T cell zone. We have studies the immune system with a focus on immune cell trafficking in various RGS deficient mice. We have supplemented these studies with in vitro studies using mRNA knock-downs as well as over expression studies using RGS proteins fused to a fluorescent marker. The recognition of the importance of both G-protein signaling and RGS proteins in the regulation of lymphocyte responses to chemokines has led us some of our studies to hematopoietic cell types. RGS1 has been linked to type 1 diabetes, celiac disease, multiple sclerosis, and melanoma progression. A prominent site of Rgs1 expression is in intraepithelial lymphocytes (IELs), which are found in the gastrointestinal tract (GI). IEL dysfunction has also been implicated in inflammatory diseases of the GI tract. In conjunction with Dr. Adrian Hayday (Kings College London Medical School) RGS1 was found to be selectively expressed at a much higher level in human gastrointestinal T cells in comparison to peripheral blood T cells, which was exaggerated in intestinal inflammation. RGS1 gain-of-function profoundly reduced T cell migration to specific lymphoid-homing chemokines, without significantly affecting random chemokinesis. Conversely, RGS1 loss-of-function enhanced directional chemotaxis, specifically in gut T cells. In addition loss of RGS1 in gut T cells reduced the severity of colitis in a mouse model. RGS2 has been linked to hypertension and anxiety disorders and mice lacking Rgs2 exhibit reduced T cell proliferation and IL-2 production, which translates in an impaired antiviral immunity in vivo. Rgs2 is well expressed in B and T lymphocytes and neutrophils while high levels are found in mast and dendritic cells. We have recently acquired Rgs2-/- mice to analyze neutrophil and B cell function in these mice. RGS10 is among the best expressed RGS proteins found in numerous hematopoietic cell types. However, immune phenotyping of these mice has revealed surprisingly little. Recently we have found that these mice have T cells with ehanced intracellular calcium responses following CD3 crosslinking. In addition patch clamping of T cells from these mice has revealed that CD3 stimulation results in a prolonged activation of CRAC channels in these mice. RGS13 is strongly expressed in both human and mouse germinal center B cells and is often found highly expressed in human B cell lymphomas of germinal center origin. Rgs13 is also well expressed in mast cells and in adult T cell leukemia cells. To facilitate our studies of RGS13 and to provide a potential resource for in vivo imaging endogenous germinal center B cells we have generated mice where GFP (green fluorescent protein) has been knocked-in (KI) into the Rgs13 locus replacing the RGS13 coding sequence. Flow cytometry revealed high levels of GFP expression in germinal center centroblasts and centrocytes. Intravital microscopy documented the presence of brightly GFP positive cells in the interfollicular region and at the T-B border within a day of immunization. Later strong expression is observed in B cells located in germinal centers. Immune phenotyping of these mice revealed a minor defect in early B cell development with a normal peripheral lymphoid compartment with the exception of a 25% expansion in the number of Peyers patches. Upon immunization the Rgs13-/- mice exhibited an enhanced extra-follicular antibody response, an exaggerated germinal center response, and a disturbance of germinal center architecture. Analysis of mRNA expression in Rgs13GFP positive B cells revealed a marked enrichment in germinal center specific genes as well as genes involved in cell proliferation. Analysis of irradiated mice reconstituted with 1:1 mix of wild type and Rgs13-/- bone marrow revealed an expansion of Rgs13-/- GC B cells at the expense of wild type cells. We are currently analyzing gene expression in germinal center B cells from these chimeric mice. These studies are aimed at understanding the role RGS13 plays in germinal center B cell function. Rgs14 is well expressed in lymphocytes and initially targeting in mice indicated that its absence resulted in embryonic lethality at the two cell stage. To analyze the role of Rgs14 in immune cells we created Rgs14LoxP mice. However, deletion of Rgs14 was not deleterious so we have analyzed mice globally lacking Rgs14 expression. Initial analysis of the immune system of these mice was unrevealing. More recent studies indicate an impaired antibody response. We are currently investigating the response of these mice to several defined antigens. Rgs19 is well expressed in B lymphocytes and less so in T cells. It is also well expressed in stem cells and hematopoietic progenitors. GFP has been inserted into the RGS19 locus and appropriated targeted mice identified. Flow cytometry revealed that GFP is well expressed in mature B and T cells and in progenitors in the bone marrow. Consistently the Rgs19 GFP KI mice had an enlarged spleen and thymus compared to wild type mice. Chemotaxis experiments comparing wild type and RGS19-/- lymphocytes indicate significant increases to a panel of chemokines with the KO B cells. Serum levels of IgM, IgG2a, and IgG2b are elevated in these mice. Current studies are aimed understanding the pathogenesis of the splenomegaly and enhanced B cell responsiveness in the Rgs19-/- mice. In bone marrow mixed chimeras the Rgs19-/- deficient B cells and neutrophils expand preferentially. Intriguing the wild type cells also expand in the mixed chimera mice suggesting that some bone marrow derived Rgs19-/- cell is responsible for the expansion. As another approach to assessing the role of RGS proteins in these cells we have begun the analysis of mice with a Gnai2 G184S knock-in obtained from Richard Neubig (University of Michigan). This mutation renders Gi alpha2 resistant to the effect of RGS proteins. The Gnai2 G184S KI mice possess a striking phenotype verifying the overall important of RGS proteins in G-protein regulation. They have a marked reduction in peripheral T cells; an increase in mature T cells in the thymus; a thymocyte egress defect; an absence of many peripheral lymph node; a disorganized spleen architecture; splenomegaly; abnormal lymphocyte responses to chemokines; and reduced serum levels of IgG3, but increased levels of Ig2b. In a thioglycolllate-induced peritonitis model, recruitment of G184S knock-in neutrophils to peritoneum is strikingly hindered despite increased numbers of neutrophils in bone marrow and spleen. In contrast, a higher number of G184S knock-in large peritoneal macrophages and CD5 negative B cells are present in the peritoneal cavity. A major chore will be to sort out which RGS proteins are responsible for the various defects in the immune and inflammatory cells in this mouse.

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。趋化因子受体主要通过触发G α i核苷酸交换来传递信号。人类和小鼠有三种Gi亚型，尽管Gi alpha2（由Gnai2编码）和Gi alpha3（由Gnai3编码）在淋巴样细胞中占主导地位。我们发现Gnai2 -/- T细胞和B细胞在趋化因子受体信号传导方面存在严重缺陷，而Gnai2 +/- T细胞和B细胞表现出适度缺陷。在体内，Gnai2-/- B细胞不能正常进入淋巴结滤泡，Gnai2-/- T细胞不能正常进入T细胞区。我们研究了免疫系统，重点是各种RGS缺陷小鼠的免疫细胞运输。我们用mRNA敲除的体外研究和RGS蛋白与荧光标记融合的过表达研究来补充这些研究。认识到g蛋白信号和RGS蛋白在调节淋巴细胞对趋化因子的反应中的重要性，使我们对造血细胞类型进行了一些研究。