SNTS--DEVELOPMENTAL FUNCTIONS AND RECEPTOR INTERACTIONS

描述(摘自申请者摘要)：SNT(Suc1结合神经营养因子诱导的酪氨酸磷酸化靶标)蛋白是最近发现的接头分子，在生长因子(包括神经营养因子(NGF、BDNF、NT3)和FGFs的刺激下发生快速酪氨酸磷酸化，但不受胰岛素和EGF的刺激。他们是美国国税局的远亲。SNTs是肉豆蔻酰化和膜锚定的，带有一个N末端的磷酸酪氨酸结合(PTB)结构域，该结构域介导着弱的但功能上重要的SNT-受体相互作用。SNTS的配体依赖的酪氨酸磷酸化使SNTS能够与至少两个不同的含有SH2结构域的部分相互作用，Grb2/SOS和Shp2。由于Grb2/SOS的激活和随后的RAS激活以及Shp2磷酸酶被证明是由成纤维细胞生长因子和神经营养素诱导的广泛的生物学反应所必需的，SNTS可能是激活两条生物关键途径所必需的。在这一应用中，提出了三个目的来阐明SNTS的生化和生物学特性。目的1利用核磁共振和诱变相结合的方法来确定SNT PTB结构域之间相互作用的性质。SNTS和另一个受体酪氨酸激酶Ret之间可能的直接相互作用也将被测试。在具体目标2中，将讨论SNT1和SNT2的生物学功能。为此将采取两种办法：1.SNTS的显性负性突变体将在培养细胞中过表达。将产生SNT基因零突变、亚型突变和条件性零突变的小鼠。最后，在具体目标3中，将检验SNTS作为潜在生物特异性决定因素的想法。为此，将生产嵌合的SNT-IRS以及胰岛素受体-Trk杂交体，以确定它们是否可以将胰岛素反应转变为神经营养因子或FGFs的反应。

DESCRIPTION (Adapted from applicant's abstract): SNT (Suc1-binding Neurotrophic factor-induced Tyrosine-phosphorylated target) proteins are recently discovered adaptor molecules that undergo rapid tyrosine phosphorylation following stimulation by growth factors including neurotrophins (NGF, BDNF, NT3) and FGFs but not by insulin and EGF. They are distantly related to IRS. SNTs are myristoylated and membrane-anchored and bear an N-terminal phosphotyrosine-binding (PTB) domain that mediates weak, but functionally significant SNT-receptor interactions. The ligand- dependent tyrosine phosphorylation of SNTs enables SNTs to interact with at least two different SH2 domain-containing moieties, Grb2/Sos and Shp2. As activation of both the Grb2/Sos and the subsequent Ras activation as well as the Shp2 phosphatase has been shown to be required for a wide-range of FGF- and neurotrophin- induced biological responses, SNTs may be required for activation of two biologically critical pathways. Three aims are proposed in this application to elucidate the biochemical and biological properties of SNTs. Aim 1 is proposed to determine the nature of the interaction between SNT PTB domains by using a combination of NMR and mutagenesis approaches. A possible direct interaction between SNTs and another receptor tyrosine kinase Ret will also be tested. In specific aim 2, the biological function of SNT1 and SNT2 will be addressed. Two approaches will be undertaken for this purpose: 1.) Dominant negative mutants of SNTs will be overexpressed in cultured cells and 2.) Mice with null, hypmerorphs and conditional null mutations in SNT genes will be generated. Finally, in specific aim 3, the idea of SNTs serving as potential biological specificity determinants will be tested. To this end, chimeric SNT-IRS as well as insulin receptor-Trk hybrids will be produced to determine if they can turn insulin response to that of neurotrophins or FGFs.