MOLECULAR MECHANISMS IN POLARIZED CELL MIGRATION

极化细胞迁移的分子机制

• 批准号: 7361400
• 负责人: JOSE VAZQUEZ-PRADO
• 金额: $ 4.74万
• 依托单位: CINVESTAV-IPN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7361400
• 关键词: ADRBK1 gene; Address; Agonist; Angiogenesis Inhibitors; Architecture; Binding; CXCR; CXCR4 gene; Cells; Complex; DNA; Elements; Embryonic Development; Endothelial Cells; Exhibits; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glutathione S-Transferase; Goals; Guanine; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Health; Human; IL8 gene; IL8RA gene; IL8RB gene; Immune response; Infection; Knowledge; Link; Mediating; Modeling; Molecular; Monitor; Movement; Neoplasm Metastasis; Participant; Peptides; Process; Properdin; Proteins; Proteomics; Recombinants; Role; Signal Transduction; Testing; Trefoil Motif; Wound Healing; Yeasts; abstracting; angiogenesis; axon growth; axonal guidance; cell motility; design; interest; migration; mutant; neoplastic cell; novel; phosducin-like protein; polarized cell; prevent; protein protein interaction; rac GTP-Binding Proteins; receptor; research study; response; rho GTP-Binding Proteins; tool; tumor; yeast two hybrid system

Revised Abstract: DESCRIPTION (provided by applicant): Polarized cell migration (PCM) is key for cell defense against infections, wound healing, axonal growth, embryonic development, tumor cell metastasis and angiogenesis. Our hypothesis postulates that P-REX1, an exchange factor for Rac, establishes critical protein-protein interactions with receptors, G proteins and novel elements participating in PCM. Our specific aims are: 1) To define molecular links leading to the activation of Rac by G proteins. 2) To determine if P-REX1, identified as a Rac activator responding to Gbetagamma and PI3K (Welch et al, 2002, Cell 108:809), establishes protein interactions relevant for PCM. 3) To determine the role of G betagamma- and P-REXl-interacting proteins in the activity of Rac and PCM. Our studies consider the modular architecture exhibited by P-REX1, which contains two DEP and two PDZ domains, suggestive of modulation by direct protein-protein interactions. Our long term goal is to understand the molecular aspects of signal transduction required for PCM of endothelial cells and to identify molecular antiangiogenic tools. The role of Gbetagamma in PCM will be monitored in the presence of phosducin-like protein- and GRK2- derived peptides. P-REX1 deletion mutants lacking the different structural domains will be prepared to identify the structural domains in P-REX1 recognized by Gbetagamma and PI3K and to reveal their role in the activation of Rac and PCM. To identify novel elements able to modulate endothelial cell migration, Gbetagamma- and P-REXl interacting proteins will be cloned by yeast two hybrid and, in parallel, will be identified by proteomic approaches. It will be determined if CXCR1 and CXCR2 GPCRs containing PDZ-interacting motives in their carboxyl terminal domain are able to establish stable interactions with P-REX1. CXCR4 that does not contain that motif will be used as a control. Human HEK293T cells will be the model to study molecular interactions. Those cells will be transfected with the diverse DNA constructs. Recombinant molecules will be expressed fused to GST, EGFP, Myc or HA tags that will facilitate their study. Endothelial cells will be used to determine the role of Gbetagamma, P-REX1 and their interacting proteins, on the activation of Rac and polarized migration responding to the activation of angiogenic G protein coupled receptors. Discovery of molecular elements that might impede unwanted endothelial cell migration will provide elements for the design of antiangiogenic treatments.

修订摘要：描述（申请人提供）：极化细胞迁移（PCM）是针对感染，伤口愈合，轴突生长，胚胎发育，肿瘤细胞转移和血管生成的细胞防御的关键。我们的假设假设P-Rex1是RAC的交换因子，它与受体，G蛋白和参与PCM的新元素建立了关键的蛋白质 - 蛋白质相互作用。我们的具体目的是：1）定义分子链接，导致G蛋白激活RAC。 2）确定p-rex1是否被确定为对Gbetagamma和PI3K响应的RAC激活剂（Welch等，2002，Cell 108：809）是否建立了与PCM相关的蛋白质相互作用。 3）确定G betagamma和p-Rexl相互作用蛋白在RAC和PCM活性中的作用。我们的研究考虑了P-Rex1展出的模块化结构，其中包含两个DEP和两个PDZ结构域，这表明直接蛋白质 - 蛋白质相互作用调节。我们的长期目标是了解内皮细胞PCM所需的信号转导的分子方面，并鉴定分子抗血管生成工具。在存在磷布斯素样蛋白和GRK2衍生肽的情况下，将监测GBETAGAMMA在PCM中的作用。缺乏不同结构结构域的P-REX1缺失突变体将准备识别Gbetagamma和PI3K识别的P-REX1中的结构域，并揭示其在RAC和PCM激活中的作用。为了识别能够调节内皮细胞迁移的新元素，将通过酵母两杂种克隆Gbetagamma和P-Rexl相互作用的蛋白质，并同时通过蛋白质组学方法鉴定。将确定是否能够在其羧基终端结构域中含有PDZ相互作用动机的CXCR1和CXCR2 GPCR能够与P-Rex1建立稳定的相互作用。不包含该基序的CXCR4将用作对照。人HEK293T细胞将成为研究分子相互作用的模型。这些细胞将用不同的DNA构建体转染。重组分子将与GST，EGFP，MYC或HA标签融合，以促进他们的研究。内皮细胞将用于确定Gbetagamma，P-Rex1及其相互作用蛋白在RAC激活和极化迁移对血管生成G蛋白偶联受体激活响应的作用。发现可能阻碍有害内皮细胞迁移的分子元素将为抗血管生成治疗的设计提供元素。

项目成果

Chimeric G alpha i2/G alpha 13 proteins reveal the structural requirements for the binding and activation of the RGS-like (RGL)-containing Rho guanine nucleotide exchange factors (GEFs) by G alpha 13.

嵌合 G α i2/G α 13 蛋白揭示了 G α 13 结合和激活含有 RGS 样 (RGL) 的 Rho 鸟嘌呤核苷酸交换因子 (GEF) 的结构要求。

• DOI: 10.1074/jbc.m410594200
• 发表时间: 2004
• 期刊: The Journal of biological chemistry
• 作者: Vázquez-Prado,José;Miyazaki,Hiroshi;Castellone,MariaDomenica;Teramoto,Hidemi;Gutkind,JSilvio
• 通讯作者: Gutkind,JSilvio

Calcium-sensing receptor endocytosis links extracellular calcium signaling to parathyroid hormone-related peptide secretion via a Rab11a-dependent and AMSH-sensitive mechanism.

钙敏感受体内吞作用通过 Rab11a 依赖性和 AMSH 敏感机制将细胞外钙信号传导与甲状旁腺激素相关肽分泌联系起来。

• DOI: 10.1210/me.2006-0523
• 发表时间: 2007
• 期刊: Molecular endocrinology (Baltimore, Md.)
• 作者: Reyes-Ibarra,AlmaP;García-Regalado,Alejandro;Ramírez-Rangel,Iliana;Esparza-Silva,AnaL;Valadez-Sánchez,Margarita;Vázquez-Prado,José;Reyes-Cruz,Guadalupe
• 通讯作者: Reyes-Cruz,Guadalupe

Modular architecture and novel protein-protein interactions regulating the RGS-containing Rho guanine nucleotide exchange factors.

调节含有 RGS 的 Rho 鸟嘌呤核苷酸交换因子的模块化结构和新型蛋白质-蛋白质相互作用。

• DOI: 10.1016/s0076-6879(04)90017-1
• 发表时间: 2004
• 期刊: Methods in enzymology
• 作者: Vazquez-Prado,Jose;Basile,John;Gutkind,JSilvio
• 通讯作者: Gutkind,JSilvio

Differential inhibitor of Gbetagamma signaling to AKT and ERK derived from phosducin-like protein: effect on sphingosine 1-phosphate-induced endothelial cell migration and in vitro angiogenesis.

来自磷酸蛋白样蛋白的 AKT 和 ERK 信号传导的 Gbetagamma 信号差异抑制剂：对 1-磷酸鞘氨醇诱导的内皮细胞迁移和体外血管生成的影响。

• DOI: 10.1074/jbc.m109.008839
• 发表时间: 2009
• 期刊: The Journal of biological chemistry
• 作者: Guzmán-Hernández,MaríaLuisa;Vázquez-Macías,Aleida;Carretero-Ortega,Jorge;Hernández-García,Ricardo;García-Regalado,Alejandro;Hernández-Negrete,Ivette;Reyes-Cruz,Guadalupe;Gutkind,JSilvio;Vázquez-Prado,José
• 通讯作者: Vázquez-Prado,José