神经营养因子受体下游信号转导通路的酵母双杂交研究
摘要
神经营养因子是指机体产生的能够促进中枢和外周神经细胞存活、生长和分化的多肽生长因子。神经营养因子既可以影响发育早期胚胎神经元或者前体细胞的生长与分化，还能够促进成熟神经细胞的存活与生长，而且它对于治疗一些神经系统退行性疾病或促进神经损伤后修复具有非常重要的作用。
神经营养因子结合并作用于神经营养因子受体，激活胞内信号通路后，才能发挥其重要的生物学效应。神经营养因子以其化学组成和受体的特性等可以分为多个家族，如GDNF家族、NGF家族等。GDNF、NGF、EGF的生物学效应分别由GDNF受体RET、NGF受体TrkA及EGF受体EGFR所介导。尽管上述配体不尽相同，但它们的受体均属于受体酪氨酸激酶(RTK)。近几年来，为了更多地理解为何不同的配体、受体可引起类似或特定的生物学效应，人们对于细胞内由这些受体介导多种信号通路的研究越来越重视。大量工作表明，配体与受体结合，引起受体的二聚化并同时激活受体后，受体可结合大量的信号蛋白分子，如接头蛋白、入坞蛋白或各种调节蛋白等。正是由于大量不同信号分子不断整合的结果，多种不同的信号得以正确传递。发现并了解这些参与受体下游通路转导的信号分对于人们更好地理解胞内信号通路无疑具有重要的意义。
因此，本研究应用酵母双杂交筛选文库的方法，寻找胞浆内神经营养因子受体酪氨酸激酶的可能底物或调控蛋白，为进一步探索受体下游的具体信号转导或调节机制打下基础。
本研究的主要结果如下：
1、将RET胞内域与LexA蛋白融合构建成pGilda-RET~(IC)，确认无自激活作用后，将其作
第H军医大学博士学位论文 口摘要（中英文）
为诱饵蛋白，经酵母双杂交方法筛选人脑LexA cDNA文库，获得274个克隆。经p－半乳糖
苦酶活性鉴定和测序分析进一步鉴别，筛选到21个质粒片段，从中获得并明确为SHZE等
基因片段。将pGilda－RET厂与pB42ADS 书共转化HLYSIg，共转化子的 p－半乳糖昔酶活
力检测呈阳性，表明在酵母中S＊2丑与RE丫”之间可相互作用。在获得稳定转染M T与＊F＊。
质粒的PC 12细胞基础上，再瞬时转染野生型SHZE或者其突变体R555E于此稳定转染细胞
株中。细胞裂解物的免疫共沉淀实验结果显示，仅当 GDNF刺激转染了野生型SHZ－B的 PC
细胞时，可以检测到RET与SHZE的结合。此结果表明，GDNF刺激过量表达RET、GFRa
与 SHZ－B的PC细胞后，SHZ－B可与 MT在体内发生结合。细胞的形态观察显示，GDNF
作用于仅转染RET与 GFRa质粒的PC细胞或转染MT、GFRa与 SHZ8质粒的PC细胞后，
细胞的分化均较明显；而作用于转染RET、GFRa与SHZE突变体R555E质粒的PC12细胞后，
分化程度明显降低，具有统计学意义。上述结果表明，MT与SHZE的结合可能参与GDNF
刺激引起PC细胞分化的信号通路。
2、将EGFR胞内域与LexA蛋白融合构建成pGilda七GFR＼确认无自激活作用后，
将其作为诱饵蛋白，经酵母双杂交方法筛选人脑 LexA cDNA文库，获得 112个克隆。经
p－半乳糖昔酶活性鉴定和测序分析进一步鉴别，筛选到6个质粒片段，从中获得并明确为
doki等基因片段。将 pGilda－EGFR’C与 pB42AD－doki共转化 HLY819，共转化子的p－半乳
糖昔酶活力测定呈阳性，表明在酵母中 doki与 EGFR‘C可结合。pGildUEGFR’C与
pB42AD刁ok1PTB酵母共转化子的p－半乳糖昔酶活力以及 SD Gal Ura“His”Tp’Leu”平板生
长实验均呈阳性，表明 doki可经 PTB结构域与 EGFR’C结合。缺失 PTB结构域的 doki门
doki A PTB）与EGFR‘’共转化子的p－半乳糖昔酶活力以及SD Ga1Ura”His’切“Leu平板生
长实验均呈阴性，从而进一步从反面证实doki可经PTB结构域与EGFR‘“结合。兔疫共沉
3
— —
淀的实验结果，进一步证实了在酵母中d。hi可经PTB结构域与EGFRIC相互作用。根据
doki PTB结构域与来自EGFR小肽的立体结构模拟提示，我们选择了d。k1PTB结构域内的
八个氨基酸残基进行突变，即73Y、74T、76L、77R、79Y、92R、93R与119I，每个氨基
酸残基均突变为丙氨酸 Ala，再将这些 dok1PTB突变体与 EGFR’“共转化酵母 HLY819，检
测共转化子的－半乳糖昔酶活力。结果显示，当PTB结构域内的73Y、76L、77R或92R
突变为川a后，EGFR’”不能与 doklPTB结合；而 74T。79Y、93R与 119I突变为 Ala后，
EGFR厂仍可与 d。klPTB结合。上述结果表明，d。k1PTB结构域内的 73Y、76L、77R和
92R残基在EGFR与doki的结合中具有重要作用。
3、将TrkA胞内域与LexA蛋白融合构建成pGilda－Ti＊，确认无自激活作用后，将其
Neurotrophic factors is a family of secreted polypeptide growth factors which could promote the development and survival of certain neuronal populations both in the peripheral and in the central nervous system. It is a target-derived polypeptide, essential for development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons as well as adult neurons in the brain. Some of the neurotrophic factors may be therapeutically beneficial for patients with nerve injury or some neurological disorders such as Alzheimer's disease and Parkinson's disease.
The biological effects of neurotrophic factors are mediated by their high-affinity ligand-receptor which present on the surface of those responsive neurons. According to the distinct components of receptors and different chemeical properties of ligands, neurotrophic factors could be classified to several families, for example GDNF and NGF family. The biological effects of GDNF, NGF and EGF were mediated by their corresponding receptor RET, TrkA and EGFR respectively. Although the ligands are totally different, all of these receptors belong to the family of receptor tyrosine kinases(RTKs). After binding with ligands, the RTKs would dimerize, simultaneously autophosphorylated and activated and then trigger the downstream signaling pathway. Since distinct signal transduction pathways are determined by the specific interaction between RTKs and their downstream effectors or regulators, it will be a possibly good strategy to investigate on the downstream molecules to better understand the
complexities of receptor signaling networks.
In present study, the human brain cDNA library was screened by using yeast two-hybrid system to search for new intracellular substrates or regulatory proteins of RET,EGFR and TrkA.
The main results of our research are as follows:
1. The intracellular part of RET receptor was fused to LexA and used as a bait to screen a human brain LexA two-hybrid cDNA library, and 274 positive clones were obtained. Among them, 21 plamids were selected and identified to be SH2-B and some other gene fragments. The interaction between RET and SH2-B was confirmed by using p-galactosidase activity assay in yeast. When overexpressed in PC 12 cells, wild type SH2-B could co-immunoprecipitated with RET in response to GDNF while SH2-B mutant R555E could not. It was observed that overexpression of wild type SH2-B enhanced GDNF-induced neurite growth while overexpression of SH2-B mutant R555E could inhibit it. Our results suggest that SH2-B could interact with RET in PC 12 cells stimulated with GDNF, and the interaction may be involved with the differentiation of PC 12 cells induced by GDNF.
2. The intracellular part of EGFR was fused to LexA and used as a bait to screen a human brain LexA two-hybrid cDNA library, and 112 positive clones were obtained. Among them, 6 plamids were selected and identified to be dokl and some other gene fragments. The interaction between EGFR and dokl was confirmed by using p-galactosidase activity assay in yeast. Moreover, we found that the interaction of EGFR with dokl was mediated by the PTB domain since the dokl PTB could bind with EGFR while the dokl A PTB could not. After alanine mutagenesis scanning study, together with yeast two-hybrid analysis, our results demonstrated that the amino acid residue 73 Y, 76L, 77R and 92R in the dokl PTB domain may be critical for
the interactaction of doklwith and EGFR.
3. The intracellular part of TrkA was fused to LexA and used as a bait to screen a human brain LexA two-hybrid cDNA library, and 269 positive clones were obtained. Among them, 11 plamids were selected and identified to be TrkAlc-BPl and some other gene fragments. The interaction between TrkAIC-BPl and TrkAIC was confirmed by using p-galactosidase activity
assay and co-immunoprecipitation experiment in yeast. Our results suggest that TrkA -BP1 is a new TrkA-binding protein and it may be a new member of rho-GAP family since the protein sequence analysis revealed that it contains a
神经营养因子结合并作用于神经营养因子受体，激活胞内信号通路后，才能发挥其重要的生物学效应。神经营养因子以其化学组成和受体的特性等可以分为多个家族，如GDNF家族、NGF家族等。GDNF、NGF、EGF的生物学效应分别由GDNF受体RET、NGF受体TrkA及EGF受体EGFR所介导。尽管上述配体不尽相同，但它们的受体均属于受体酪氨酸激酶(RTK)。近几年来，为了更多地理解为何不同的配体、受体可引起类似或特定的生物学效应，人们对于细胞内由这些受体介导多种信号通路的研究越来越重视。大量工作表明，配体与受体结合，引起受体的二聚化并同时激活受体后，受体可结合大量的信号蛋白分子，如接头蛋白、入坞蛋白或各种调节蛋白等。正是由于大量不同信号分子不断整合的结果，多种不同的信号得以正确传递。发现并了解这些参与受体下游通路转导的信号分对于人们更好地理解胞内信号通路无疑具有重要的意义。
因此，本研究应用酵母双杂交筛选文库的方法，寻找胞浆内神经营养因子受体酪氨酸激酶的可能底物或调控蛋白，为进一步探索受体下游的具体信号转导或调节机制打下基础。
本研究的主要结果如下：
1、将RET胞内域与LexA蛋白融合构建成pGilda-RET~(IC)，确认无自激活作用后，将其作
第H军医大学博士学位论文 口摘要（中英文）
为诱饵蛋白，经酵母双杂交方法筛选人脑LexA cDNA文库，获得274个克隆。经p－半乳糖
苦酶活性鉴定和测序分析进一步鉴别，筛选到21个质粒片段，从中获得并明确为SHZE等
基因片段。将pGilda－RET厂与pB42ADS 书共转化HLYSIg，共转化子的 p－半乳糖昔酶活
力检测呈阳性，表明在酵母中S＊2丑与RE丫”之间可相互作用。在获得稳定转染M T与＊F＊。
质粒的PC 12细胞基础上，再瞬时转染野生型SHZE或者其突变体R555E于此稳定转染细胞
株中。细胞裂解物的免疫共沉淀实验结果显示，仅当 GDNF刺激转染了野生型SHZ－B的 PC
细胞时，可以检测到RET与SHZE的结合。此结果表明，GDNF刺激过量表达RET、GFRa
与 SHZ－B的PC细胞后，SHZ－B可与 MT在体内发生结合。细胞的形态观察显示，GDNF
作用于仅转染RET与 GFRa质粒的PC细胞或转染MT、GFRa与 SHZ8质粒的PC细胞后，
细胞的分化均较明显；而作用于转染RET、GFRa与SHZE突变体R555E质粒的PC12细胞后，
分化程度明显降低，具有统计学意义。上述结果表明，MT与SHZE的结合可能参与GDNF
刺激引起PC细胞分化的信号通路。
2、将EGFR胞内域与LexA蛋白融合构建成pGilda七GFR＼确认无自激活作用后，
将其作为诱饵蛋白，经酵母双杂交方法筛选人脑 LexA cDNA文库，获得 112个克隆。经
p－半乳糖昔酶活性鉴定和测序分析进一步鉴别，筛选到6个质粒片段，从中获得并明确为
doki等基因片段。将 pGilda－EGFR’C与 pB42AD－doki共转化 HLY819，共转化子的p－半乳
糖昔酶活力测定呈阳性，表明在酵母中 doki与 EGFR‘C可结合。pGildUEGFR’C与
pB42AD刁ok1PTB酵母共转化子的p－半乳糖昔酶活力以及 SD Gal Ura“His”Tp’Leu”平板生
长实验均呈阳性，表明 doki可经 PTB结构域与 EGFR’C结合。缺失 PTB结构域的 doki门
doki A PTB）与EGFR‘’共转化子的p－半乳糖昔酶活力以及SD Ga1Ura”His’切“Leu平板生
长实验均呈阴性，从而进一步从反面证实doki可经PTB结构域与EGFR‘“结合。兔疫共沉
3
— —
淀的实验结果，进一步证实了在酵母中d。hi可经PTB结构域与EGFRIC相互作用。根据
doki PTB结构域与来自EGFR小肽的立体结构模拟提示，我们选择了d。k1PTB结构域内的
八个氨基酸残基进行突变，即73Y、74T、76L、77R、79Y、92R、93R与119I，每个氨基
酸残基均突变为丙氨酸 Ala，再将这些 dok1PTB突变体与 EGFR’“共转化酵母 HLY819，检
测共转化子的－半乳糖昔酶活力。结果显示，当PTB结构域内的73Y、76L、77R或92R
突变为川a后，EGFR’”不能与 doklPTB结合；而 74T。79Y、93R与 119I突变为 Ala后，
EGFR厂仍可与 d。klPTB结合。上述结果表明，d。k1PTB结构域内的 73Y、76L、77R和
92R残基在EGFR与doki的结合中具有重要作用。
3、将TrkA胞内域与LexA蛋白融合构建成pGilda－Ti＊，确认无自激活作用后，将其
Neurotrophic factors is a family of secreted polypeptide growth factors which could promote the development and survival of certain neuronal populations both in the peripheral and in the central nervous system. It is a target-derived polypeptide, essential for development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons as well as adult neurons in the brain. Some of the neurotrophic factors may be therapeutically beneficial for patients with nerve injury or some neurological disorders such as Alzheimer's disease and Parkinson's disease.
The biological effects of neurotrophic factors are mediated by their high-affinity ligand-receptor which present on the surface of those responsive neurons. According to the distinct components of receptors and different chemeical properties of ligands, neurotrophic factors could be classified to several families, for example GDNF and NGF family. The biological effects of GDNF, NGF and EGF were mediated by their corresponding receptor RET, TrkA and EGFR respectively. Although the ligands are totally different, all of these receptors belong to the family of receptor tyrosine kinases(RTKs). After binding with ligands, the RTKs would dimerize, simultaneously autophosphorylated and activated and then trigger the downstream signaling pathway. Since distinct signal transduction pathways are determined by the specific interaction between RTKs and their downstream effectors or regulators, it will be a possibly good strategy to investigate on the downstream molecules to better understand the
complexities of receptor signaling networks.
In present study, the human brain cDNA library was screened by using yeast two-hybrid system to search for new intracellular substrates or regulatory proteins of RET,EGFR and TrkA.
The main results of our research are as follows:
1. The intracellular part of RET receptor was fused to LexA and used as a bait to screen a human brain LexA two-hybrid cDNA library, and 274 positive clones were obtained. Among them, 21 plamids were selected and identified to be SH2-B and some other gene fragments. The interaction between RET and SH2-B was confirmed by using p-galactosidase activity assay in yeast. When overexpressed in PC 12 cells, wild type SH2-B could co-immunoprecipitated with RET in response to GDNF while SH2-B mutant R555E could not. It was observed that overexpression of wild type SH2-B enhanced GDNF-induced neurite growth while overexpression of SH2-B mutant R555E could inhibit it. Our results suggest that SH2-B could interact with RET in PC 12 cells stimulated with GDNF, and the interaction may be involved with the differentiation of PC 12 cells induced by GDNF.
2. The intracellular part of EGFR was fused to LexA and used as a bait to screen a human brain LexA two-hybrid cDNA library, and 112 positive clones were obtained. Among them, 6 plamids were selected and identified to be dokl and some other gene fragments. The interaction between EGFR and dokl was confirmed by using p-galactosidase activity assay in yeast. Moreover, we found that the interaction of EGFR with dokl was mediated by the PTB domain since the dokl PTB could bind with EGFR while the dokl A PTB could not. After alanine mutagenesis scanning study, together with yeast two-hybrid analysis, our results demonstrated that the amino acid residue 73 Y, 76L, 77R and 92R in the dokl PTB domain may be critical for
the interactaction of doklwith and EGFR.
3. The intracellular part of TrkA was fused to LexA and used as a bait to screen a human brain LexA two-hybrid cDNA library, and 269 positive clones were obtained. Among them, 11 plamids were selected and identified to be TrkAlc-BPl and some other gene fragments. The interaction between TrkAIC-BPl and TrkAIC was confirmed by using p-galactosidase activity
assay and co-immunoprecipitation experiment in yeast. Our results suggest that TrkA -BP1 is a new TrkA-binding protein and it may be a new member of rho-GAP family since the protein sequence analysis revealed that it contains a
引文
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Ogawa W et al. Evidence for two distinct 60-kilodalton substrates of the SRC tyrosine kinase. J Biol Chem 1994, 269: 29602-29608.
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Price JV et al. The maternal ventralizing locus torpedo is allelic to faint little ball, an embryonic lethal, and encodes the Drosophila EGF receptor homolog. Cell 1989, 56:1085-1092.
Qian X et al. Identifacation and characterization of novel substrates of Trk receptors in developing neurons.Neuron 1998,21:1017-1029.
Qian X et al. SH2-B and APS are multimeric adapters that augment TrkA signaling.Mol Cell Biol2001,21(5):1613-1620.
Riedel H et al. PSM, an insulin-dependent, pro-rich, PH, SH2 domain containing partner of the insulin receptor. J Biochem 1997,122:1105-1113.
Rommel C et al. Ras-a versatile cellular switch. Curr Opin Genet Dev 1998, 8:412-418.
Rui L et al. Identification of SH2-B beta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling.Mol Cell Biol 1997,6633-6644.
Rui L et al. Platelet-derived growth factor(PDGF) stimulates the association of SH2-B beta with PDGF receptor and phosphorylation of SH2-B beta. J Biol Chem 1998,273:21239-21245.
Rui L et al. SH2-B Is Required for Nerve Growth Factor-induced Neuronal Differentiation. J biol Chem 1999, 274(15): 10590-10594.
Rui L et al. Identification of SH2-B beta as a potent cytoplasmic activator of the tyrosine kinase Janus kinase 2.Proc Natl Acad Sci USA 1999, 96:7172-7177.
Schejter ED et al. The Drosophila EGF receptor homolog (DER) gene is allelic to faint little ball, a locus essential for embryonic development. Cell 1989, 56:1093-1104.
Sibilia M et al. Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 1995, 269:234-238.
Sternberg PW et al. Conspiracy Theory: Ras and Raf do not act alone. Cell 1998, 95:447-450.
Suzu S et al. p56(dok-2) as a cytokine-inducible inhibitor of cell proliferation and signal transduction. EMBO J 2000,19:5114-5122.
Tamir I et al. The RasGAP-binding protein p62dok is a mediator of inhibitory FcgammaRIIB signals in B cells. Immunity 2000,12:347-358.
Tessier-Lavigne M et al. The molecular biology of axon guidence. Science 1996,274:1123-1133.
Threadgill DW et al. Targeted disruption of mouse EGF receptor-effect of genetic background on mutant phenotype. Science 1995,269:230-234.
Ullrich A and Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell 1990, 61:203-212.
van der Geer P et al. Receptor protein-tyrosine kinases and-their signal transduction pathways. Annu Rev Cell Biol 1994,10:251-337.
van Dijk TB et al. Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells. Blood 2000, 96(10):3406-3413.
Yamada M et al. The neurotrophic action and signalling of epidermal growth factor. Prog Neurobiol 1997 Jan; 51(1): 19-37.
Yamanashi Y et al. Identification of the Abl-and rasGAP-associated 62kDa protein as a docking protein, Dok. Cell 1997, 88: 205-211.
Yamanashi Y et al. Role of the rasGAP-associated docking protein p62(dok) in negative regulation of B cell receptor-mediated signaling. Genes Dev 2000,14,: 11-16.
Yamashita H et al. The Csk homologous kinase associates with TrkA receptors and is involved in neurite outgrowth of PC12 cells. J Biol Chem 1999,274:15059-15065.
Yokouchi M et al. Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation. Oncogene 1997,15:7-15.
Zhang B et al. The insulin receptor-related receptor. Tissue expression, ligand binding specificity, and signaling capabilities. J Biol Chem 1992,67(26): 18320-18328.