SCIRR10蛋白及其相关研究
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摘要
脊髓损伤修复相关10号基因(spinal cord injury and regeneration related gene No 10, SCIRR10)是我们实验室从大鼠脊髓原代神经元损伤消减杂交文库中筛选到的一个基因。前期研究发现SCIRR10基因分布于几乎整个中枢神经系统,多种技术证明了SCIRR10参与脊髓损伤与修复,发现损伤后脊髓内SCIRR10 mRNA和蛋白表达明显高调;通过酵母双杂交技术,发现了SCIRR10可以与促甲状腺素释放激素受体2(TRH-R2)和羧肽酶E(CPE)相互作用;通过多种实验证实了TRH-R2是SCIRR10蛋白的结合受体:信号转导研究发现SCIRR10蛋白可激活TRH-R2介导的MAPK-ERK和磷酸脂酶C两个信号转导通路,这些激活作用可以分别被PTX、U73122或U0126阻断;功能研究实验显示,SCIRR10是一个新型神经营养因子,痕量的SCIRR10体外可明显促进原代皮层神经元的存活;SCIRR10具有强大的促进皮层和脊髓组织块神经再生作用,体内研究显示了SCIRR10促进大鼠损伤坐骨神经的再生作用。本文一方面探讨SCIRR10蛋白功能作用位点,通过实验验证这些位点在与TRH-R2相互作用中起到关键作用,并且激活下游的信号通路;另一方面进一步证实SCIRR10与CPE的相互作用。
本文的研究主要分为以下三个部分:
第一部分:SCIRR10蛋白与TRH-R2作用位点的确定。通过对SCIRR10蛋白及其同源序列的计算,寻找出该蛋白存在3个作用位点:F_(46)Y_(54)Y_(64),F_(80)Y_(81)Y_(87),F_(128)Y_(132)Y_(136)。为了探讨SCIRR10蛋白位点作用方式,一方面将SCIRR10蛋白从C端开始依次进行截短,然后验证SCIRR10蛋白片段是否能与TRH-R2结合;另一方面是将SCIRR10蛋白的3个作用位点部分单独分离出来,并且将所有的苯丙氨酸(F)和酪氨酸(Y)突变为亮氨酸(L)或将苯丙氨酸突变为亮氨酸、酪氨酸突变为苯丙氨酸,然后验证正常的或者突变的作用位点短肽是否能与TRH-R2结合。
1、应用PCR扩增技术、真/原核载体构建技术、GST-pull down技术、western blot技术,验证了3个从C端开始依次截短的SCIRR10蛋白片段都能够与TRH-R2相互结合。初步证明了SCIRR10蛋白3个作用位点全部或部分参与了TRH-R2的相互作用。
2、荧光共振能量转移技术验证截短的SCIRR10蛋白片段和突变体是否在细胞体内与TRH-R2产生FRET现象。采集的图像数据通过统计学方法处理。实验证明,包含有F46Y54Y64位点的截短SCIRR10蛋白片段能够在细胞内与TRH-R2相互作用。其它的SCIRR10短肽与TRH-R2不能产生FRET现象。F_(46)Y_(54)Y_(64)是SCIRR10与TRH-R2关键结合部位(位点)。
3、通过GFP和RFP双色荧光标记蛋白技术确定SCIRR10短肽与TRH-R2在细胞的定位分布。首先获得含有SCIRR10短肽的细胞培养基上清,将其作用于表达TRH-R2的COS-7细胞,通过激光共聚焦显微镜观察二者在细胞中的分布。包含有F_(46)Y_(54)Y_(64)位点的SCIRR10短肽与TRH-R2蛋白在细胞膜上出现了红绿荧光相互重叠的现象,其它实验组没有观察到这种现象的发生。该实验间接地证明前一个FRET实验的结果,可以说明F_(46)Y_(54)Y_(64)位点在SCIRR10与TRH-R2相互识别中起关键作用。
第二部分:SCIRR10短肽对TRH-R2介导的下游信号通路作用。以PC12细胞为研究材料,检测SCIRR10短肽是否能够如同SCIRR10全长蛋白一样,刺激TRH-R2介导的下游信号通路。
1、通过RT-PCR和Western blot两种不同的方法,分别从转录水平和翻译水平证明了TRH-R2存在于PC12细胞株。
2、应用Western blot技术检测MAPK信号通路中的ERK1/2和Elk-1磷酸化现象的发生。SCIRR10短肽对PC12细胞进行刺激处理,包含有F_(46)Y_(54)Y_(64)位点的SCIRR10短肽能够如同SCIRR10全长和TRH一样,诱导MAPK信号通路中ERK1/2和Elk-1磷酸化水平变化。其它的实验组和阴性组却不能产生明显的变化。
第三部分:SCIRR10蛋白及CPE相互作用验证。通过前期酵母双杂交技术筛选,了解到CPE能够与SCIRR10作用。为了进一步了解SCIRR10和CPE之间的相互作用。对二者进行了深入的研究。
1、通过RT-PCR的方法,从大鼠脑cDNA文库中获取CPE基因全长,然后将其克隆到真核表达载体pcDNA3.1/myc-His中。应用Western blot方法检测CPE蛋白的表达情况。大鼠CPE蛋白可以在COS-7细胞中得以正确表达。
2、研究CPE蛋白是否能够在体外与SCIRR10蛋白相互作用。首先构建了CPE原核表达载体,然后通过Pull-Down方法,证明CPE蛋白在体外能够与CPE相互结合。
3、荧光能量转移技术验证SCIRR10蛋白和CPE蛋白是否在细胞中相互作用。基于Sensitized Emmission方法,验证了SCIRR10能够在细胞内与CPE相互作用。采集的图像数据通过统计学方法处理,证明SCIRR10与CPE实验组产生的FRET现象高于阴性对照组。
4、通过免疫荧光染色的方法验证SCIRR10蛋白和CPE蛋白在细胞中的定位。SCIRR10蛋白能够与Golgi体重叠标记;在SCIRR10和CPE共转染的COS-7细胞中,SCIRR10蛋白和CPE蛋白重叠标记。
综上所述,SCIRR10蛋白中的F_(46)Y_(54)Y_(64)位点是与TRHR2相互作用的关键部位。包含有F_(46)Y_(54)Y_(64)位点的SCIRR10蛋白片段能够激活TRH-R2介导的下游信号通路变化。SCIRR10蛋白与CPE蛋白的相互作用通过FRET和GST pull-down得到了进一步验证,在细胞体内和体外,二者都存在相互作用。SCIRR10蛋白在Golgi体合成加工后,能够与CPE相互结合,说明CPE在SCIRR10蛋白运转过程中起到重要作用。
Spinal cord injury and regeneration related gene No 10 (SCIRR10) is a gene in our effort of seeking genes responsive to spinal cord neuron injury. Preliminary studies revealed that the gene located in almost the entire central nervous system. The relationship between SCIRR10 and spinal cord injury was proved by in situ hybridization, RT-PCR and Western Blot. SCIRR10 mRNA and protein expression were significantly upgraded after the spinal cord was injured. It was found SCIRR10 had interaction with thyrotropin-releasing hormone receptor 2 (TRH-R2) and carboxypeptidase E (CPE) by yeast two-hybrid technology. The interaction between SCIRR10 and receptor TRH-R2 was proved by protein Pull-Down technique, and TRH-R2 was confirmed as a receptor of SCIRR10 fatherly by using of ligand - receptor interaction technique. SCIRR10 could activate MAPK-ERK and phospholipase C signal transduction pathways. Activities of SCIRR10 were blocked by PTX, U73122 or U0126. SCIRR10 protein could significantly promote the survival of primary cortical neurons in vitro. SCIRR10 was a powerful promotion of the cortex and spinal cord nerve tissue regeneration. The purpose of this paper is reveal the SCIRR10 protein site of binding with TRH-R2. On the other hand, the interaction of SCIRR10 and CPE was confirmed.
This paper mostly includes the following three parts: Part I: The functional site of SCIRR10 protein which bind with TRH-R2 was identified. SCIRR10 protein sequence was simulated and analysised by computer. SCIRR10 protein contains three sites of function as follows:F_(46)Y_(54)Y_(64); F80Y81Y87; F128Y132Y136. Truncated SCIRR10 protein which had one,two or three sites were tested. On the other hand, peptide of mutations were tested also. F and Y was replaced by L or F was replaced by L and Y was replaced by F in the mutations of SCIRR10 protein fragments.
1. Three truncated SCIRR10 protein were detected by GST-pull down and western blot. It was confirmed that three different truncated protein of SCIRR10 could bind with TRH-R2 in vitro.
2. Truncated SCIRR10 protein and mutations were detected by fluorescence resonance energy transfer. The date of image which were collected to deal with statistics methods. It was confirmed truncated SCIRR10 protein which had F_(46)Y_(54)Y_(64) could produce phenomenon of fluorescence resonance energy transfer with TRH-R2 in vivo. On the contrary, other groups couldn’t produce the phenomenon. The site of F_(46)Y_(54)Y_(64) is important in the binding with TRH-R2.
3. The localization of truncated SCIRR10 protein fragments and TRH-R2 in cells were deteceted by two Colour Fluorescence labeling protein technology. TRH-R2 was translated to the COS-7 cells. Expression of TRH-R2 was observed after translated 24h. The cells was treated with supernatant which had truncated SCIRR10 protein fragments. Truncated SCIRR10 protein which had F_(46)Y_(54)Y_(64) could observe green fluorescence overlaying on the red fluorescence at the same cell. The site of F_(46)Y_(54)Y_(64), should be playing an crucial role in the interaction of SCIRR10
and TRH-R2. Part II: TRH-R2 mediated signaling pathways which were simulated by SCIRR10 protein fragments were detected. Truncated SCIRR10 protein fragments and mutations were used to stimulated the PC12 cells, and detected changes of TRH-R2 mediated signaling pathway by western blot.
1. Expression of TRH-R2 gene on the PC12 cells was confirmed by RT-PCR and western blot.
2. The PC12 cells was treated with supernatant which had truncated SCIRR10 protein fragments and mutations. The change of ERK1/2 phosphorylation and Elk-1 phosphorylation was detected by western blot. Only truncated SCIRR10 protein which had F_(46)Y_(54)Y_(64) could cause changes. F_(46)Y_(54)Y_(64) of SCIRR10 should be the function section.
Part III: The interaction of SCIRR10 and CPE was confirmed. In early experiments, Carboxypeptidase E(CPE) and thyrotropin releasing hormone receptor 2(TRH-R2) were screened for proteins interaction with SCIRR10 by yeast two-hybrid technique. CPE is an enzyme which participates in the process of biosynthesis of neurotransmitter. The interaction between CPE and SCIRR10 should be tested by further experiments.
1. CPE gene was cloned from rat brain cDNA library by RT-PCR. CPE gene was cloned into the vector pcDNA3.1/myc-His. CPE gene was expressed successfully on the COS-7 cells.
2. The interaction of SCIRR10 and CPE was detected in vivo by fluorescence energy transfer technology. FRET efficiency was collected by Sensitized Emmission method. The image date was deal with statistical method. The FRET efficiency of SCIRR10 and CPE group far more than the negative control group.
3. The protein-protein interaction of SCIRR10 and CPE in intro was detected. The prokaryotic expression vector of CPE was constructed at the beginning. The combination of SCIRR10 and CPE in vitro was confirmed by pull-down method.
4.The position of SCIRR10 and CPE were identified in the cells by immunofluorescence staining method. SCIRR10 protein could overlap with the Golgi body. SCIRR10 protein and CPE protein overlapped at the cells which were cotransfected with SCIRR10 and CPE.
In summary, F_(46)Y_(54)Y_(64) is the key binding site of SCIRR10 and THR-R2. The SCIRR10 protein fragments which contain F_(46)Y_(54)Y_(64) can activate TRH-R2-mediated the signaling pathways. The protein-protein interaction of SCIRR10 and CPE has been furtherly validated in vivo and in vitro. SCIRR10 protein was processed in the Golgi at first. SCIRR10 can be combined with CPE then. CPE is an important role during the process of SCIRR10.
本文的研究主要分为以下三个部分:
第一部分:SCIRR10蛋白与TRH-R2作用位点的确定。通过对SCIRR10蛋白及其同源序列的计算,寻找出该蛋白存在3个作用位点:F_(46)Y_(54)Y_(64),F_(80)Y_(81)Y_(87),F_(128)Y_(132)Y_(136)。为了探讨SCIRR10蛋白位点作用方式,一方面将SCIRR10蛋白从C端开始依次进行截短,然后验证SCIRR10蛋白片段是否能与TRH-R2结合;另一方面是将SCIRR10蛋白的3个作用位点部分单独分离出来,并且将所有的苯丙氨酸(F)和酪氨酸(Y)突变为亮氨酸(L)或将苯丙氨酸突变为亮氨酸、酪氨酸突变为苯丙氨酸,然后验证正常的或者突变的作用位点短肽是否能与TRH-R2结合。
1、应用PCR扩增技术、真/原核载体构建技术、GST-pull down技术、western blot技术,验证了3个从C端开始依次截短的SCIRR10蛋白片段都能够与TRH-R2相互结合。初步证明了SCIRR10蛋白3个作用位点全部或部分参与了TRH-R2的相互作用。
2、荧光共振能量转移技术验证截短的SCIRR10蛋白片段和突变体是否在细胞体内与TRH-R2产生FRET现象。采集的图像数据通过统计学方法处理。实验证明,包含有F46Y54Y64位点的截短SCIRR10蛋白片段能够在细胞内与TRH-R2相互作用。其它的SCIRR10短肽与TRH-R2不能产生FRET现象。F_(46)Y_(54)Y_(64)是SCIRR10与TRH-R2关键结合部位(位点)。
3、通过GFP和RFP双色荧光标记蛋白技术确定SCIRR10短肽与TRH-R2在细胞的定位分布。首先获得含有SCIRR10短肽的细胞培养基上清,将其作用于表达TRH-R2的COS-7细胞,通过激光共聚焦显微镜观察二者在细胞中的分布。包含有F_(46)Y_(54)Y_(64)位点的SCIRR10短肽与TRH-R2蛋白在细胞膜上出现了红绿荧光相互重叠的现象,其它实验组没有观察到这种现象的发生。该实验间接地证明前一个FRET实验的结果,可以说明F_(46)Y_(54)Y_(64)位点在SCIRR10与TRH-R2相互识别中起关键作用。
第二部分:SCIRR10短肽对TRH-R2介导的下游信号通路作用。以PC12细胞为研究材料,检测SCIRR10短肽是否能够如同SCIRR10全长蛋白一样,刺激TRH-R2介导的下游信号通路。
1、通过RT-PCR和Western blot两种不同的方法,分别从转录水平和翻译水平证明了TRH-R2存在于PC12细胞株。
2、应用Western blot技术检测MAPK信号通路中的ERK1/2和Elk-1磷酸化现象的发生。SCIRR10短肽对PC12细胞进行刺激处理,包含有F_(46)Y_(54)Y_(64)位点的SCIRR10短肽能够如同SCIRR10全长和TRH一样,诱导MAPK信号通路中ERK1/2和Elk-1磷酸化水平变化。其它的实验组和阴性组却不能产生明显的变化。
第三部分:SCIRR10蛋白及CPE相互作用验证。通过前期酵母双杂交技术筛选,了解到CPE能够与SCIRR10作用。为了进一步了解SCIRR10和CPE之间的相互作用。对二者进行了深入的研究。
1、通过RT-PCR的方法,从大鼠脑cDNA文库中获取CPE基因全长,然后将其克隆到真核表达载体pcDNA3.1/myc-His中。应用Western blot方法检测CPE蛋白的表达情况。大鼠CPE蛋白可以在COS-7细胞中得以正确表达。
2、研究CPE蛋白是否能够在体外与SCIRR10蛋白相互作用。首先构建了CPE原核表达载体,然后通过Pull-Down方法,证明CPE蛋白在体外能够与CPE相互结合。
3、荧光能量转移技术验证SCIRR10蛋白和CPE蛋白是否在细胞中相互作用。基于Sensitized Emmission方法,验证了SCIRR10能够在细胞内与CPE相互作用。采集的图像数据通过统计学方法处理,证明SCIRR10与CPE实验组产生的FRET现象高于阴性对照组。
4、通过免疫荧光染色的方法验证SCIRR10蛋白和CPE蛋白在细胞中的定位。SCIRR10蛋白能够与Golgi体重叠标记;在SCIRR10和CPE共转染的COS-7细胞中,SCIRR10蛋白和CPE蛋白重叠标记。
综上所述,SCIRR10蛋白中的F_(46)Y_(54)Y_(64)位点是与TRHR2相互作用的关键部位。包含有F_(46)Y_(54)Y_(64)位点的SCIRR10蛋白片段能够激活TRH-R2介导的下游信号通路变化。SCIRR10蛋白与CPE蛋白的相互作用通过FRET和GST pull-down得到了进一步验证,在细胞体内和体外,二者都存在相互作用。SCIRR10蛋白在Golgi体合成加工后,能够与CPE相互结合,说明CPE在SCIRR10蛋白运转过程中起到重要作用。
Spinal cord injury and regeneration related gene No 10 (SCIRR10) is a gene in our effort of seeking genes responsive to spinal cord neuron injury. Preliminary studies revealed that the gene located in almost the entire central nervous system. The relationship between SCIRR10 and spinal cord injury was proved by in situ hybridization, RT-PCR and Western Blot. SCIRR10 mRNA and protein expression were significantly upgraded after the spinal cord was injured. It was found SCIRR10 had interaction with thyrotropin-releasing hormone receptor 2 (TRH-R2) and carboxypeptidase E (CPE) by yeast two-hybrid technology. The interaction between SCIRR10 and receptor TRH-R2 was proved by protein Pull-Down technique, and TRH-R2 was confirmed as a receptor of SCIRR10 fatherly by using of ligand - receptor interaction technique. SCIRR10 could activate MAPK-ERK and phospholipase C signal transduction pathways. Activities of SCIRR10 were blocked by PTX, U73122 or U0126. SCIRR10 protein could significantly promote the survival of primary cortical neurons in vitro. SCIRR10 was a powerful promotion of the cortex and spinal cord nerve tissue regeneration. The purpose of this paper is reveal the SCIRR10 protein site of binding with TRH-R2. On the other hand, the interaction of SCIRR10 and CPE was confirmed.
This paper mostly includes the following three parts: Part I: The functional site of SCIRR10 protein which bind with TRH-R2 was identified. SCIRR10 protein sequence was simulated and analysised by computer. SCIRR10 protein contains three sites of function as follows:F_(46)Y_(54)Y_(64); F80Y81Y87; F128Y132Y136. Truncated SCIRR10 protein which had one,two or three sites were tested. On the other hand, peptide of mutations were tested also. F and Y was replaced by L or F was replaced by L and Y was replaced by F in the mutations of SCIRR10 protein fragments.
1. Three truncated SCIRR10 protein were detected by GST-pull down and western blot. It was confirmed that three different truncated protein of SCIRR10 could bind with TRH-R2 in vitro.
2. Truncated SCIRR10 protein and mutations were detected by fluorescence resonance energy transfer. The date of image which were collected to deal with statistics methods. It was confirmed truncated SCIRR10 protein which had F_(46)Y_(54)Y_(64) could produce phenomenon of fluorescence resonance energy transfer with TRH-R2 in vivo. On the contrary, other groups couldn’t produce the phenomenon. The site of F_(46)Y_(54)Y_(64) is important in the binding with TRH-R2.
3. The localization of truncated SCIRR10 protein fragments and TRH-R2 in cells were deteceted by two Colour Fluorescence labeling protein technology. TRH-R2 was translated to the COS-7 cells. Expression of TRH-R2 was observed after translated 24h. The cells was treated with supernatant which had truncated SCIRR10 protein fragments. Truncated SCIRR10 protein which had F_(46)Y_(54)Y_(64) could observe green fluorescence overlaying on the red fluorescence at the same cell. The site of F_(46)Y_(54)Y_(64), should be playing an crucial role in the interaction of SCIRR10
and TRH-R2. Part II: TRH-R2 mediated signaling pathways which were simulated by SCIRR10 protein fragments were detected. Truncated SCIRR10 protein fragments and mutations were used to stimulated the PC12 cells, and detected changes of TRH-R2 mediated signaling pathway by western blot.
1. Expression of TRH-R2 gene on the PC12 cells was confirmed by RT-PCR and western blot.
2. The PC12 cells was treated with supernatant which had truncated SCIRR10 protein fragments and mutations. The change of ERK1/2 phosphorylation and Elk-1 phosphorylation was detected by western blot. Only truncated SCIRR10 protein which had F_(46)Y_(54)Y_(64) could cause changes. F_(46)Y_(54)Y_(64) of SCIRR10 should be the function section.
Part III: The interaction of SCIRR10 and CPE was confirmed. In early experiments, Carboxypeptidase E(CPE) and thyrotropin releasing hormone receptor 2(TRH-R2) were screened for proteins interaction with SCIRR10 by yeast two-hybrid technique. CPE is an enzyme which participates in the process of biosynthesis of neurotransmitter. The interaction between CPE and SCIRR10 should be tested by further experiments.
1. CPE gene was cloned from rat brain cDNA library by RT-PCR. CPE gene was cloned into the vector pcDNA3.1/myc-His. CPE gene was expressed successfully on the COS-7 cells.
2. The interaction of SCIRR10 and CPE was detected in vivo by fluorescence energy transfer technology. FRET efficiency was collected by Sensitized Emmission method. The image date was deal with statistical method. The FRET efficiency of SCIRR10 and CPE group far more than the negative control group.
3. The protein-protein interaction of SCIRR10 and CPE in intro was detected. The prokaryotic expression vector of CPE was constructed at the beginning. The combination of SCIRR10 and CPE in vitro was confirmed by pull-down method.
4.The position of SCIRR10 and CPE were identified in the cells by immunofluorescence staining method. SCIRR10 protein could overlap with the Golgi body. SCIRR10 protein and CPE protein overlapped at the cells which were cotransfected with SCIRR10 and CPE.
In summary, F_(46)Y_(54)Y_(64) is the key binding site of SCIRR10 and THR-R2. The SCIRR10 protein fragments which contain F_(46)Y_(54)Y_(64) can activate TRH-R2-mediated the signaling pathways. The protein-protein interaction of SCIRR10 and CPE has been furtherly validated in vivo and in vitro. SCIRR10 protein was processed in the Golgi at first. SCIRR10 can be combined with CPE then. CPE is an important role during the process of SCIRR10.
引文
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[4]Kimura I, Konishi M, Asaki T, et al. Neudesin, an extracellular heme-binding protein, suppresses adipogenesis in 3T3-L1 cells via the MAPK cascade. Biochem Biophys Res Commun. 2009 Mar 27;381(1):75-80.
[5] Cao J, O’Donnell D, Vu H, Payza K et al. Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. Journal of Biological Chemistry 1998 273 32281–32287.
[6] Itadani H, Nakamura T, Itoh J et al. Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochemical and Biophysical Research Communications 1998 250 68–71.
[7] O’Dowd BF, Lee DK, Huang W et al. TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1. Molecular Endocrinology 2000 14 183–193.
[8] Harder S, Lu X, Wang W et al. Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2). Endocrinology 2001 142 1188–1194.
[9] Fricker LD. Carboxypeptidase E. Annu Rev Physiol. 1988, 50 :309-321.
[10]Maccumber MW, Snyder SH, Ross CA. Carboxypeptidase E(Enkephalin Convertase): Mrna Distribution in Rat Brain by in situ Hybridization. J Neurosci. 1990,10(8):2850-2860.
[11] Lou H, Kim SK, Zaitsev E, et al. Sorting and Activity-Dependent Secretion of BDNF Require Interaction of a Specific Motif with the Sorting Receptor Carboxypeptidade E. Neuron. 2005, 45(2):245-255.
[12] Wu CM, Chang HT, Chang MD. Membrane-bound carboxypeptidade E facilitates the entry of eosinophil cationic protein into neuroendocrine cells. Biochem. J. 2004,382(Pt 3):841-848
[1] Kimura I, Yoshioka M, Konishi M, et al. Neudesin, a novel secreted protein with a unique primary structure and neurotrophic activity. J Neurosci Res. 2005 79(3):287-94.
[2]Kimura I, Konishi M, Miyake A, et al. Neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells. J Neurosci Res. 2006 83(8):1415-24
[3]Kimura I, Nakayama Y, Yamauchi H, et al. Neurotrophic activity of neudesin, a novel extracellular heme-binding protein, is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain. J Biol Chem. 2008 283(7):4323-31.
[4]Kimura I, Konishi M, Asaki T, et al. Neudesin, an extracellular heme-binding protein, suppresses adipogenesis in 3T3-L1 cells via the MAPK cascade. Biochem Biophys Res Commun. 2009 Mar 27;381(1):75-80.
[5] Cao J, O’Donnell D, Vu H, Payza K et al. Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. Journal of Biological Chemistry 1998 273 32281–32287.
[6] Itadani H, Nakamura T, Itoh J et al. Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochemical and Biophysical Research Communications 1998 250 68–71.
[7] O’Dowd BF, Lee DK, Huang W et al. TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1. Molecular Endocrinology 2000 14 183–193.
[8] Harder S, Lu X, Wang W et al. Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2). Endocrinology 2001 142 1188–1194.
[1] de la Pena P, Delgado LM, del Camino D et al. Two isoforms of the thyrotropin-releasing hormone receptor generated by alternative splicing have indistinguishable functional properties. Journal of Biological Chemistry 267 25703–25708.
[2] Zhao D, Yang J, Jones KE, Gerald C et al. Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells. [erratum appears in Endocrinology 1993 132 2658]. Endocrinology 130 3529–3536.
[3] Sellar RE, Taylor PL, Lamb RF et al. Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland. Journal of Molecular Endocrinology 1993 10 199–206.
[4] Sun YM, Millar RP, Ho H et al. Cloning and characterization of the chicken thyrotropin-releasing hormone receptor. Endocrinology 1998 139 3390–3398.
[5] Takata M, Shimada Y, Ikeda A et al. Molecular cloning of bovine thyrotropin-releasing hormone receptor gene. Journal of Veterinary Medical Science 1998 60 123–127.
[6] Harder S, Dammann O, Buck F et al. Cloning of two thyrotropin-releasing hormone receptor subtypes from a lower vertebrate (Catostomus commersoni):functional expression, gene structure, and evolution. General and Comparative Endocrinology 2001 124 236–245.
[7] Duthie SM, Taylor PL, Anderson L et al. Cloning and functional characterisation of the human TRH receptor. Molecular Cell Endocrinology 1993 95 R11–R15.
[8] Matre V, Karlsen HE, Wright MS et al. Molecular cloning of a functional human thyrotropin-releasing hormone receptor. Biochemical and Biophysical Research Communications 1993 195 179–185.
[9] Cao J, O’Donnell D, Vu H, Payza K et al.1998 Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. Journal of Biological Chemistry 1998 273 32281–32287.
[10] Itadani H, Nakamura T, Itoh J et al. Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochemical and Biophysical Research Communications 1998 250 68–71.
[11] O’Dowd BF, Lee DK, Huang W et al. TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1. Molecular Endocrinology 2000 14 183–193.
[12] Harder S, Lu X, Wang W et al. Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2). Endocrinology 2001 142 1188–1194.
[13] Sun Y, Lu X, Gershengorn MC Thyrotropin-releasing hormone receptors -- similarities and differences. J Mol Endocrinol. 2003 Apr; 30(2):87-97.
[14]赵勇,张远强,张金山促甲状腺激素释放激素受体-2在大鼠生后睾丸不同发育阶段的表达和定位<<解剖学报>>2005年第36卷第04期
[15] Zhao Y, Hou WG, Zhu HP et al. Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells. Cell Tissue Res. 2008 Nov; 334(2):283-94.
[16]Hsieh KP & Martin TF Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipaseC by coupling to the guanosine triphosphate-binding proteins Gq and G11. 1992. Molecular Endocrinology 6 1673–1681.
[17]Kiley SC, Parker PJ, Fabbro D et al. Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4C1 cells. Journal of Biological Chemistry 1991 266 23761–23768.
[18]Jefferson AB, Travis SM & Schulma Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells. Journal of Biological Chemistry 1991 266 1484–1490.
[19]Cui ZJ, Gorelick FS & Dannies PS Calcium/calmodulindependent protein kinase-II activation in rat pituitary cells in the presence of thyrotropin-releasing hormone and dopamine. Endocrinology 1994 134 2245–2250.
[20]Kanda Y, Koike K, Ohmichi M, Sawada T, Hirota K et al. A possible involvement of Yosine kinase in TRH-induced prolactin secretion in GH3 cells. Biochemical and Biophysical Research Communications 1994. 199 1447–1452
[21]Ohmichi M, Sawada T, Kanda Y, Koike K, Hirota K, Miyake A et al. Thyrotropin-releasing hormone stimulates MAP kinase activity in GH3 cells by divergent pathways. Evidence of a role for early Yosine phosphorylation. Journal of Biological Chemistry 1994. 269 3783–3788.
[22]Gershengorn MC & Osman R Molecular and cellular biology of thyrotropin-releasing hormone receptors. Physiological Reviews 1996. 76 175–191.
[23]Curran T, Rauscher FJ 3rd, Cohen DR & Franza BR Jr Beyond the second messenger: oncogenes and transcription factors. Cold Spring Harbour Symposium of Quantitative Biology 1988. 53 769–777.
[24]Andrisani OM CREB-mediated transcriptional control. Critical Reviews in Eukaryotic GeneExpression1999 9 19–32.
[25]Gutkind JS The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. Journal of Biological Chemistry 1998. 273 1839–1842.
[26]Wang W & Gershengorn MC Rat TRH receptor type 2 exhibits higher basal signaling activity than TRH receptor type 1.Endocrinology 1999. 140 4916–4919.
[27]Sun Y & Gershengorn MC Correlation between basal signaling and internalization of thyrotropin-releasing hormone receptors: evidence for involvement of similar receptor conformations. Endocrinology 2002 .143 2886–2892.
[1] Fricker LD. Carboxypeptidase E. Annu Rev Physiol. 1988, 50 :309-321.
[2]Maccumber MW, Snyder SH, Ross CA. Carboxypeptidase E(Enkephalin Convertase): Mrna Distribution in Rat Brain by in situ Hybridization. J Neurosci. 1990,10(8):2850-2860.
[3]Fricker LD, Devi L. Posttranslational processing of carboxypeptidase E, a neuropeptiade-processing enzyme, in AtT-20 cells and bovine pituitary secretory granules. J Neurochem. 1993, 61(4):1404-1415.
[4]Hook VY. Differential distribution of carboxypeptiadase-processing enzyme activity and immunoreactivity in membrane and soluble components of chromaffin granules. J Neurochem. 1985. 45(3):987-989.
[5]Cool DR, Normant E, Shen F, et al. Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpe(fat)mice. Cell.1997,88(1):73-83.
[6]Shen FS, Loh YP. Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in cpe(fat) mice associated with a carboxypeptidase E mutation. Proc Natl Acad Sci USA.1997, 94(10):5314-5319.
[7]Zhang CF, Snell CR, Loh YP. Identification of a novel prohormone sorting signal-binding site oncarboxypeptidase E, a regulated secretory pathway-sorting receptor. Mol Endocrinol. 1999,13(4):527-536.
[8] Lou H, Kim SK, Zaitsev E, et al. Sorting and Activity-Dependent Secretion of BDNF Require Interaction of a Specific Motif with the Sorting Receptor Carboxypeptidade E. Neuron. 2005, 45(2):245-255.
[9] Wu CM, Chang HT, Chang MD. Membrane-bound carboxypeptidade E facilitates the entry of eosinophil cationic protein into neuroendocrine cells. Biochem. J. 2004,382(Pt 3):841-848
[10]Zhou A, Minami M, Zhu X, et al. Altered biosynthesis of neuropeptide processing enzyme carboxypeptidase E after brain ischemia : molecular mechanism and implication. J Cerb Blood Flow Metab. 2004,24(6): 612-622
[1] de la Pena P, Delgado LM, del Camino D et al. Two isoforms of the thyrotropin-releasing hormone receptor generated by alternative splicing have indistinguishable functional properties. Journal of Biological Chemistry 267 25703–25708.
[2] Zhao D, Yang J, Jones KE, Gerald C et al. Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells. [erratum appears in Endocrinology 1993 132 2658]. Endocrinology 130 3529–3536.
[3] Sellar RE, Taylor PL, Lamb RF et al. Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland. Journal of Molecular Endocrinology 1993 10 199–206.
[4] Sun YM, Millar RP, Ho H et al. Cloning and characterization of the chicken thyrotropin-releasing hormone receptor. Endocrinology 1998 139 3390–3398.
[5] Takata M, Shimada Y, Ikeda A et al. Molecular cloning of bovine thyrotropin-releasing hormone receptor gene. Journal of Veterinary Medical Science 1998 60 123–127.
[6] Harder S, Dammann O, Buck F et al. Cloning of two thyrotropin-releasing hormone receptor subtypes from a lower vertebrate (Catostomus commersoni):functional expression, gene structure, and evolution. General and Comparative Endocrinology 2001 124 236–245.
[7] Duthie SM, Taylor PL, Anderson L et al. Cloning and functional characterisation of the human TRH receptor. Molecular Cell Endocrinology 1993 95 R11–R15.
[8] Matre V, Karlsen HE, Wright MS et al. Molecular cloning of a functional human thyrotropin-releasing hormone receptor. Biochemical and Biophysical Research Communications 1993 195 179–185.
[9] Cao J, O’Donnell D, Vu H, Payza K et al.1998 Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. Journal of Biological Chemistry 1998 273 32281–32287.
[10] Itadani H, Nakamura T, Itoh J et al. Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochemical and Biophysical Research Communications 1998 250 68–71.
[11] O’Dowd BF, Lee DK, Huang W et al. TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1. Molecular Endocrinology 2000 14183–193.
[12] Harder S, Lu X, Wang W et al. Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2). Endocrinology 2001 142 1188–1194.
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[21] Kiley SC, Parker PJ, Fabbro D et al. Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4C1 cells. Journal of Biological Chemistry 1991 266 23761–23768.
[22] Jefferson AB, Travis SM & Schulman H et al. Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells. Journal of Biological Chemistry 1991 266 1484–1490.
[23] Cui ZJ, Gorelick FS & Dannies PS et al. Calcium/calmodulindependent protein kinase-II activation in rat pituitary cells in the presence of thyrotropin-releasing hormone and dopamine. Endocrinology 1994 134 2245–2250.
[24] Kanda Y, Koike K, Ohmichi M, et al. A possible involvement of tyrosine kinase in TRH-induced prolactin secretion in GH3 cells. Biochemical and Biophysical Research Communications 1994 199 1447–1452.
[25] Ohmichi M, Sawada T, Kanda Y, et al. Thyrotropin-releasing hormone stimulates MAP kinase activity in GH3 cells by divergent pathways. Evidence of a role for early tyrosine phosphorylation. Journal of Biological Chemistry 1994 269 3783–3788.
[26] Gershengorn MC & Osman R Molecular and cellular biology of thyrotropin-releasing hormone receptors. Physiological Reviews 1996 76 175–191.
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[31] Gutkind JS The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. Journal of Biological Chemistry 1998 273 1839–1842.
[32] Wang W & Gershengorn MC Rat TRH receptor type 2 exhibits higher basal signaling activity than TRH receptor type 1. Endocrinology 1999 140 4916–4919.
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[64]Kimura I, Nakayama Y, Yamauchi H, et al. Neurotrophic activity of neudesin, a novel extracellular heme-binding protein, is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain. J Biol Chem. 2008 Feb 15; 283(7):4323-31.
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[2]Kimura I, Konishi M, Miyake A, et al. Neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells.J Neurosci Res. 2006 83(8):1415-24
[3]Kimura I, Nakayama Y, Yamauchi H, et al. Neurotrophic activity of neudesin, a novel extracellular heme-binding protein, is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain. J Biol Chem. 2008 283(7):4323-31.
[4]Kimura I, Konishi M, Asaki T, et al. Neudesin, an extracellular heme-binding protein, suppresses adipogenesis in 3T3-L1 cells via the MAPK cascade. Biochem Biophys Res Commun. 2009 Mar 27;381(1):75-80.
[5] Cao J, O’Donnell D, Vu H, Payza K et al. Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. Journal of Biological Chemistry 1998 273 32281–32287.
[6] Itadani H, Nakamura T, Itoh J et al. Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochemical and Biophysical Research Communications 1998 250 68–71.
[7] O’Dowd BF, Lee DK, Huang W et al. TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1. Molecular Endocrinology 2000 14 183–193.
[8] Harder S, Lu X, Wang W et al. Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2). Endocrinology 2001 142 1188–1194.
[9] Fricker LD. Carboxypeptidase E. Annu Rev Physiol. 1988, 50 :309-321.
[10]Maccumber MW, Snyder SH, Ross CA. Carboxypeptidase E(Enkephalin Convertase): Mrna Distribution in Rat Brain by in situ Hybridization. J Neurosci. 1990,10(8):2850-2860.
[11] Lou H, Kim SK, Zaitsev E, et al. Sorting and Activity-Dependent Secretion of BDNF Require Interaction of a Specific Motif with the Sorting Receptor Carboxypeptidade E. Neuron. 2005, 45(2):245-255.
[12] Wu CM, Chang HT, Chang MD. Membrane-bound carboxypeptidade E facilitates the entry of eosinophil cationic protein into neuroendocrine cells. Biochem. J. 2004,382(Pt 3):841-848
[1] Kimura I, Yoshioka M, Konishi M, et al. Neudesin, a novel secreted protein with a unique primary structure and neurotrophic activity. J Neurosci Res. 2005 79(3):287-94.
[2]Kimura I, Konishi M, Miyake A, et al. Neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells. J Neurosci Res. 2006 83(8):1415-24
[3]Kimura I, Nakayama Y, Yamauchi H, et al. Neurotrophic activity of neudesin, a novel extracellular heme-binding protein, is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain. J Biol Chem. 2008 283(7):4323-31.
[4]Kimura I, Konishi M, Asaki T, et al. Neudesin, an extracellular heme-binding protein, suppresses adipogenesis in 3T3-L1 cells via the MAPK cascade. Biochem Biophys Res Commun. 2009 Mar 27;381(1):75-80.
[5] Cao J, O’Donnell D, Vu H, Payza K et al. Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. Journal of Biological Chemistry 1998 273 32281–32287.
[6] Itadani H, Nakamura T, Itoh J et al. Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochemical and Biophysical Research Communications 1998 250 68–71.
[7] O’Dowd BF, Lee DK, Huang W et al. TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1. Molecular Endocrinology 2000 14 183–193.
[8] Harder S, Lu X, Wang W et al. Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2). Endocrinology 2001 142 1188–1194.
[1] de la Pena P, Delgado LM, del Camino D et al. Two isoforms of the thyrotropin-releasing hormone receptor generated by alternative splicing have indistinguishable functional properties. Journal of Biological Chemistry 267 25703–25708.
[2] Zhao D, Yang J, Jones KE, Gerald C et al. Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells. [erratum appears in Endocrinology 1993 132 2658]. Endocrinology 130 3529–3536.
[3] Sellar RE, Taylor PL, Lamb RF et al. Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland. Journal of Molecular Endocrinology 1993 10 199–206.
[4] Sun YM, Millar RP, Ho H et al. Cloning and characterization of the chicken thyrotropin-releasing hormone receptor. Endocrinology 1998 139 3390–3398.
[5] Takata M, Shimada Y, Ikeda A et al. Molecular cloning of bovine thyrotropin-releasing hormone receptor gene. Journal of Veterinary Medical Science 1998 60 123–127.
[6] Harder S, Dammann O, Buck F et al. Cloning of two thyrotropin-releasing hormone receptor subtypes from a lower vertebrate (Catostomus commersoni):functional expression, gene structure, and evolution. General and Comparative Endocrinology 2001 124 236–245.
[7] Duthie SM, Taylor PL, Anderson L et al. Cloning and functional characterisation of the human TRH receptor. Molecular Cell Endocrinology 1993 95 R11–R15.
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[12] Harder S, Lu X, Wang W et al. Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2). Endocrinology 2001 142 1188–1194.
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[14]赵勇,张远强,张金山促甲状腺激素释放激素受体-2在大鼠生后睾丸不同发育阶段的表达和定位<<解剖学报>>2005年第36卷第04期
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