MOLECULAR MECHANISMS OF MAST CELL SECRETION--ROLE OF RAB

肥大细胞分泌的分子机制--RAB的作用

• 批准号: 2415215
• 负责人: Bridget S Wilson
• 金额: $ 10.3万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415215
• 关键词: affinity chromatography; antibody receptor; chimeric proteins; complementary DNA; crosslink; exocytosis; granule; guanine nucleotide binding protein; guanine nucleotide exchange factors; immunocytochemistry; immunoelectron microscopy; immunofluorescence technique; laboratory rat; mast cell; membrane fusion; membrane proteins; molecular cloning; nucleic acid probes; protein isoforms; protein sequence; protein tyrosine kinase; recombinant proteins; site directed mutagenesis; synthetic peptide; transfection; transfection /expression vector; affinity chromatography; antibody receptor; chimeric proteins; complementary DNA; crosslink; exocytosis; granule; guanine nucleotide binding protein; guanine nucleotide exchange factors; immunocytochemistry; immunoelectron microscopy; immunofluorescence technique; laboratory rat; mast cell; membrane fusion; membrane proteins; molecular cloning; nucleic acid probes; protein isoforms; protein sequence; protein tyrosine kinase; recombinant proteins; site directed mutagenesis; synthetic peptide; transfection; transfection /expression vector

In mast cells, basophils and the RBL-2H3 mast cell line, antigens that crosslink the high affinity IgE receptor, Fc-epsilonR1, lead to the release of histamine and other inflammatory agents by degranulation. Although receptor-mediated degranulation is GTP-dependent, the Fc- epsilonR1 is not a classical G-protein linked receptor. Rather it belongs to the family of immune system antigen receptors, including the T cell receptor, the mIg receptor of B cells and the Fc-gamma receptors, that modulate cellular function through the activation of cytoplasmic tyrosine kinases. Growth factor receptors also signal through tyrosine kinase activation; recent studies in the EGF and PDGF receptor systems have shown that the prototypic low molecular weight G-protein, ras, is activated when the autophosphorylated receptor stimulates the SH2/SH3 adaptor protein, Grb2, that complexes with the guanine nucleotide exchange factor, Sos. Rab3 proteins are ras-related proteins that are implicated in synaptic vesicle fusion and regulated exocytosis from endocrine cells. Recently, we obtained a full-length clone of a rab3 isoform, rab3D, from RBL-2H3 cells. Rab3D's hypervariable regions contain two proline-rich motifs, PXXP, that fit the criteria for binding to SH3 (Src homology 3) domains. We hypothesize that rab3D is the principal mediator of GTP-dependent, Ca2+- regulated secretion in mast cells. We hypothesize further that its activation occurs by a signaling cascade in which Fc-epsilonR1-associated tyrosine kinases activate a SH2/SH3-containing adaptor protein/guanine nucleotide exchange factor complex that in turn activates rab3D. Immunolocalization studies with rab3D-specific antibodies will be used to demonstrate the association of rab3D with RBL-2H3 cell secretory granules. The hypothesis that rab3D activity is essential for degranulation will be tested by studies of granule morphology and secretion in stable RBL-2H3 transfectants overexpressing mutant rab3D proteins that are defective in guanine nucleotide binding or GTPase activity. Rab3D mutants which lack the two PXXP motifs will be designed to test the hypothesis that these SH3-binding regions are essential to couple Fc-epsilonR1 crosslinking to secretion PXXP-motif peptides will be used as affinity supports to purify and characterize SH3 domain-containing proteins which may serve as adaptors, guanine nucleotide exchange factors, or both for rab3D. Finally, the conformationally locked rab3D mutants will be used in co-precipitation studies and as affinity substrates in order to identify proteins which interact with rab3D downstream of activation; these proteins are likely to represent components of the mast cell granule's docking or fusion machinery. The proposed studies are expected to provide a complete description of the pathway coupling Fc-epsilonR1 crosslinking to secretion in mast cells. They will serve as a model for understanding mechanisms of regulated secretion in other immune system cells.

在肥大细胞，嗜碱性粒细胞和RBL-2H3肥大细胞系，抗原 交叉链接高亲和力IgE受体FC-EPSILONR1导致 通过脱粒释放组胺和其他炎症剂。 尽管受体介导的脱粒是GTP依赖性的，但FC- Epsilonr1不是经典的G蛋白连接的受体。相反，它属于 免疫系统抗原受体的家族，包括T细胞 受体，B细胞的MIG受体和FC-GAMMA受体的受体， 通过细胞质酪氨酸的激活来调节细胞功能 激酶。生长因子受体也通过酪氨酸激酶发出信号 激活; EGF和PDGF受体系统的最新研究已显示 当原型低分子量G蛋白RAS Ras激活时，当 自磷酸化受体刺激SH2/SH3适配器蛋白， GRB2，与鸟嘌呤核苷酸交换因子相结合，SOS。 Rab3蛋白是与RAS相关的蛋白，与突触 囊泡融合和内分泌细胞的调节胞吐作用。最近，我们 从RBL-2H3细胞中获得了Rab3同工型Rab3d的全长克隆。 Rab3d的高变量区域包含两个富含脯氨酸的图案PXXP， 符合与SH3（SRC同源3）结构域结合的标准。我们 假设Rab3d是GTP依赖性Ca2+ - 的主要介体 调节肥大细胞的分泌。我们进一步假设它 激活是通过信号级联反应发生的，其中FC- epsilonr1相关 酪氨酸激酶激活含SH2/SH3的适配器蛋白/鸟嘌呤 核苷酸交换因子复合物又激活了rab3d。 RAB3D特异性抗体的免疫定位研究将用于 演示了RAB3D与RBL-2H3细胞分泌颗粒的关联。 Rab3d活性对于脱粒至关重要的假设将是 通过稳定RBL-2H3中颗粒形态和分泌的研究测试 过表达突变体Rab3d蛋白的转染剂有缺陷 鸟嘌呤核苷酸结合或GTPase活性。缺乏的rab3d突变体 两个PXXP基序将设计为测试这些假设 SH3结合区域对于夫妇fc-epsilonr1交联至关重要 分泌PXXP-MOTIF肽将用作纯化的亲和力支持 并表征含SH3域的蛋白质，可以用作 适配器，鸟嘌呤核苷酸交换因子或Rab3d的辅助因子。最后， 构象锁定的RAB3D突变体将用于共沉淀 研究和作为亲和力底物，以鉴定蛋白质 与激活下游的Rab3d相互作用；这些蛋白可能会 代表肥大细胞颗粒对接或融合的组件 机械。拟议的研究预计将提供完整的 路径耦合FC-EPSILONR1交联的说明与分泌 在肥大细胞中。他们将作为理解机制的模型 其他免疫系统细胞中的分泌。