LIGAND BINDING DOMAINS OF TGF BETA RECEPTOR

描述：（来自研究者摘要。）此应用程序的 目的是结构解释的特异性， 转化生长因子β（TGF β）与TGF β受体结合 （TbetaR）。 TGF β与II型和I型的胞外结构域结合 TbetaR. 这两种类型都是跨膜丝氨酸/苏氨酸激酶受体。 根据细胞环境，TGF β抑制或刺激细胞内的 动物和人类的分裂。 在乳腺癌，肝癌，胃癌， 结肠和皮肤肿瘤，以及B和T淋巴瘤，失活突变 在Tbeta基因的两个等位基因中， 对TGF β的敏感性允许不受限制的细胞生长和肿瘤 进展 对这些经济和/或第三产业知之甚少。 TbetaR的配体结合结构域（exTbetaR）的结构。 本申请的具体目的是： 1. 以毫克量生产15 N/13 C标记的exTbetaR II型 2. exTbetaR的1H、15 N和13 C核的序列特异性分配 II型 3. exTbetaR II型的溶液结构 4. 表面TGF β 1结合位点的实验鉴定 exTR II型 5. 以毫克量生产15 N13 C标记的exTbetaR I型 6. exTbetaR的1H、15 N和13 C核的序列特异性分配 I型 7. ExTbetaR I型的溶液结构 exT β R在大肠杆菌中表达为融合蛋白。杆菌重组 系统 亲和层析用于纯化融合蛋白 含有exT β R，然后通过亲和和尺寸排阻纯化 层析 高度优化的表达系统用于 富集纯化的蛋白质中的15 N和13 C同位素。 结构 使用NMR方法在溶液中进行exT β R的测定。 多维异谱NMR技术用于共振 构象约束的赋值和提取。 这些 约束将用于计算具有距离的3D结构 几何和能量最小化方法。 配体的结构 受体的结合域将构成研究的基础， 配体-受体相互作用 确定关键地点， 在结合模型配体时它们中发生的变化将随之发生。 这项研究将使未来的设计激动剂和拮抗剂的细胞 在动物发育、生理学和免疫学过程中的增殖和分化， 肿瘤进展。 有越来越多的已知序列， TGF β超家族蛋白质中的受体，如从大规模 排序的努力。 这项研究的长期目标是发现 确定结合位点位置和特异性的原则 新的受体，其中直接的实验方法， 配体-受体复合物不可行。

DESCRIPTION: (From the investigator's abstract.) This application's objective is the structural explanation of the specificity of the transforming growth factor beta (TGFbeta) binding to the TGFbeta receptors (TbetaR). TGFbeta is bound by exoplasmic domains of the type II and type I TbetaR. Both types are transmembrane serine/threonine kinase receptors. Depending on the cellular context, TGFbeta inhibits or stimulates cellular division in animals and humans. In breast carcinomas, hepatomas, gastric, colon and skin tumors, as well as B and T lymphomas, inactivating mutations in both alleles of the Tbeta genes occur and correlate with loss of sensitivity to TGFbeta allowing unrestrained cell growth and tumor progression. Very little is known about thesecondary and/or tertiary structures of the ligand binding domains of TbetaRs (exTbetaR). Specific aims of this application are: 1. Production of 15N/13C labeled exTbetaR type II in milligram quantities 2. Sequence specific assignments of 1H, 15N, and 13C nuclei of the exTbetaR type II 3. Solution structure of the exTbetaR type II 4. Experimental identification of the TGFbeta1 binding sites on the surface of the exTR type II 5. Production of 15N13C labeled exTbetaR type I in milligram quantities 6. Sequence specific assignments of 1H, 15N, and 13C nuclei of the exTbetaR type I 7. Solution structure of the exTbetaR type I The exTbetaRs are expressed as fusion proteins in an E. coli recombinant system. Affinity chromatography is used to purify fusion proteins containing exTbetaRs, which are then purified by affinity and size exclusion chromatography. The highly optimized expression system is used for enrichment of the purified protein in 15N and 13C isotopes. The structure determination o the exTbetaRs is carried out in solution using NMR methods. multidimensional heteronuclear NMR techniques are used for resonance assignments and extraction of the conformational constraints. These constraints will be used to calculate the 3D structure with distance geometry and energy minimization methods. The structures of the ligand binding domains of receptors will form the basis for studies of ligand-receptor interactions. Identification of the critical sites and changes occurring in them upon binding of the model ligand will follow. This research will allow future design of agonists and antagonists of cell proliferation and differentiation during animal development, physiology, and tumor progression. There is an ever increasing number of known sequences of receptors in the TGFbeta superfamily of proteins as derived from large scale sequencing efforts. The long range goal of this research is to discover the principles that determine locations of binding sites and specificities of new receptors, for which the direct experimental approach to the ligand-receptor complex is not feasible.