G蛋白偶联受体激酶5在胚胎造血过程中作用的研究及其相互作用蛋白的筛选
发布时间：2018-09-01 11:07
【摘要】：G蛋白偶联受体(G-protein-coupled receptor, GPCR)是细胞表面最大的受体超家族,它能够介导多种多样的配体信号,包括神经递质、激素、气味、光线及其它小分子,同时也介导多种药物的活性,从而调节众多的生理病理过程。G蛋白偶联受体激酶(G-protein-coupled receptor kinases, GRKs)是调节GPCR的一类关键的丝/苏氨酸激酶。当受体被活化后,GRK通过结合并迅速磷酸化受体的胞内段,抑制受体的持续激活,导致受体的内吞和脱敏。GRK有七种亚型(GRK1-7),根据其结构和功能的不同分为三个亚家族。其中GRK5是研究较多的成员之一,它广泛分布于全身多种器官,尤其高表达于心脏和肺,对多种GPCR具有选择性的调节作用,参与调节各种各样的生理病理过程。 以往对于GRK的研究主要集中于在发育成熟的正常的机体及组织中的功能和调节作用。近年来,GRKs在胚胎发育过程中的作用也越来越受到重视,有研究发现：GRK2基因缺失的小鼠胚胎表现为生长迟缓和胚胎发育停滞,并且于胚胎期E15.5天内死亡。我的硕士课题的第一部分工作以GRK5基因敲除小鼠为模型初步探索了GRK5在小鼠胚胎造血系统发育中作用及其机制；同时我们实验室以前的研究发现发现GRK5可以结合p53并通过使其磷酸化而促进p53的降解,进而影响p53依赖的细胞凋亡、细胞周期调控等重要功能；另外我们前期研究还发现GRK5在肿瘤的生长中起了重要的作用。也结合前人的研究,这都提示GRK5存在着许多未知的新功能。为此,我的硕士课题的第二部分工作通过免疫沉淀-蛋白质组的方法寻找GRK5的相互作用蛋白,为研究GRK5的新的功能研究提供新的线索。实验结果发现： 1、GRK5基因敲除小鼠胚胎血岛发育受损。我们采用了GRK5基因敲除小鼠的胚胎进行切片,并通过HE染色观察血岛的发育情况,发现与野生型胚胎相比,GRK5基因敲除的小鼠胚胎血岛发育受损,仅有较少的胚胎(约27%)可观察到正常的血岛。这提示GRK5参与小鼠胚胎造血的过程。 2、免疫共沉淀和GST-Pull Down实验结果提示,GRK5与GATA2之间存在着相互作用。在HEK293T细胞中通过免疫共沉淀实验发现GRK5与GATA2存在相互作用。接下来通过体外结合实验,我们将纯化的GST或GST-GATA2蛋白与Flag-HA-GRK5和GST偶联的琼脂糖树脂一起孵育,得到的免疫复合物用SDS-PAGE进行分离,再用Western Blot方法进行检测,发现GRK5和GATA2存在直接的相互作用。 3、从野生型及GRK5基因敲除的成年小鼠眼眶静脉丛采血后检测外周血常规,发现与野生型小鼠相比,GRK5基因敲除的成年小鼠外周血中白细胞显著减少且主要为粒细胞的减少,红细胞和血小板数目二者之间无差异。 4、通过分别在人脐静脉内皮细胞(HUVEC)和人乳腺癌细胞(MDA-MB-231)中过表达GRK5,运用免疫沉淀/质谱的方法检测了与GRK5有相互作用的蛋白,分析后结合免疫沉淀实验也对其进行了验证,我们发现,这些蛋白可大致分为三类：与RNA剪切相关的蛋白；与DNA损伤修复相关的蛋白；细胞骨架相关蛋白。这些都提示GRK5可能通过这些与其相互作用的蛋白发挥多种多样的目前还未知的功能 5、综上所述,本研究发现了GRK5在小鼠胚胎造血中的调节作用及其对成年小鼠血液系统的影响,并新发现了一些与GRK5存在相互作用的蛋白。其中,GRK5基因敲除后会导致小鼠胚胎血岛发育受损,且成年小鼠外周血中白细胞的数目显著减少,而外周血中白细胞的降低主要是粒细胞绝对数的减少。通过免疫沉淀／质谱实验,我们发现与GRK5存在相互作用的蛋白中,一部分参与RNA的剪切；一部分与DNA损伤修复相关；还有一部分属于细胞骨架相关蛋白,这些都提示GRK5可能通过这些与其相互作用的蛋白发挥多种多样的目前还未知的功能,对于研究GRK5的新功能有一定的提示作用。
[Abstract]:G-protein-coupled receptor (GPCR) is the largest receptor superfamily on the cell surface. It can mediate a variety of ligand signals, including neurotransmitters, hormones, odors, light and other small molecules. It also mediates the activities of a variety of drugs, thereby regulating a wide range of physiological and pathological processes. Protein-coupled receptor kinases (GRKs) are a key class of serine/threonine kinases that regulate GPCR. When the receptor is activated, GRK inhibits the receptor's continuous activation by binding to and rapidly phosphorylating the receptor's intracellular segment, resulting in endocytosis and desensitization of the receptor. GRK has seven subtypes (GRK1-7), which are divided into three subtypes according to their structure and function. GRK5 is one of the most widely studied members of the family. It is widely distributed in many organs of the body, especially in the heart and lungs. GRK5 selectively regulates various GPCRs and participates in various physiological and pathological processes.
In recent years, more and more attention has been paid to the role of GRKs in the process of embryonic development. Some studies have found that mouse embryos with GRK2 gene deletion exhibit growth retardation and embryonic development arrest, and at E15.5 days of embryonic development. The first part of my Master's program explored the role and mechanism of GRK5 in the development of mouse embryonic hematopoietic system by using GRK5 knockout mice as a model. At the same time, previous studies in our laboratory have found that GRK5 binds to p53 and promotes the degradation of p53 by phosphorylation, thereby affecting p53-dependent detail. In addition, our previous studies also found that GRK5 plays an important role in the growth of tumors. In combination with previous studies, this suggests that GRK5 has many unknown new functions. The interaction proteins provide new clues for the study of new functions of GRK5.
1. The development of blood islands in GRK5 knockout mice embryos was impaired. We used the embryos of GRK5 knockout mice to observe the development of blood islands by HE staining. Compared with wild type embryos, the development of blood islands in GRK5 knockout mice embryos was impaired. Only a few embryos (about 27%) could observe normal blood islands. GRK5 is involved in the process of mouse embryo hematopoiesis.
2. Immunocoprecipitation and GST-Pull Down assays suggested that there was an interaction between GRK5 and GATA2. The interaction between GRK5 and GATA2 was found in HEK293T cells by immunocoprecipitation assay. Next, the purified GST or GST-GATA2 protein was combined with flag-HA-GRK5 and GST-coupled agarose resin through in vitro binding assay. After incubation, the immune complexes were separated by SDS-PAGE and detected by Western Blot method. It was found that there was a direct interaction between GRK5 and GATA2.
3. Peripheral blood samples were collected from the orbital venous plexus of wild-type and GRK5 knockout adult mice. Compared with wild-type mice, the number of white blood cells in the peripheral blood of GRK5 knockout adult mice was significantly reduced, mainly granulocytes, and there was no difference in the number of red blood cells and platelets.
4. By overexpressing GRK5 in human umbilical vein endothelial cells (HUVEC) and human breast cancer cells (MDA-MB-231), the proteins interacting with GRK5 were detected by immunoprecipitation/mass spectrometry. The proteins were analyzed and validated by immunoprecipitation assay. We found that these proteins can be divided into three groups: they are related to RNA shearing. Proteins; proteins associated with DNA damage repair; cytoskeleton-related proteins. These suggest that GRK5 may play a variety of previously unknown functions through these proteins interacting with GRK5
5. To sum up, we have found that GRK5 regulates the hematopoiesis of mouse embryos and its effect on the blood system of adult mice. We have also found some interacting proteins with GRK5. By immunoprecipitation/mass spectrometry, we found that some of the proteins interacting with GRK5 were involved in RNA splicing, some were associated with DNA damage and repair, and some were cytoskeleton-related proteins, which suggested that GRK5 might pass through. These proteins interacting with GRK5 play a variety of unknown functions, which may be helpful for the study of new functions of GRK5.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
[Abstract]:G-protein-coupled receptor (GPCR) is the largest receptor superfamily on the cell surface. It can mediate a variety of ligand signals, including neurotransmitters, hormones, odors, light and other small molecules. It also mediates the activities of a variety of drugs, thereby regulating a wide range of physiological and pathological processes. Protein-coupled receptor kinases (GRKs) are a key class of serine/threonine kinases that regulate GPCR. When the receptor is activated, GRK inhibits the receptor's continuous activation by binding to and rapidly phosphorylating the receptor's intracellular segment, resulting in endocytosis and desensitization of the receptor. GRK has seven subtypes (GRK1-7), which are divided into three subtypes according to their structure and function. GRK5 is one of the most widely studied members of the family. It is widely distributed in many organs of the body, especially in the heart and lungs. GRK5 selectively regulates various GPCRs and participates in various physiological and pathological processes.
In recent years, more and more attention has been paid to the role of GRKs in the process of embryonic development. Some studies have found that mouse embryos with GRK2 gene deletion exhibit growth retardation and embryonic development arrest, and at E15.5 days of embryonic development. The first part of my Master's program explored the role and mechanism of GRK5 in the development of mouse embryonic hematopoietic system by using GRK5 knockout mice as a model. At the same time, previous studies in our laboratory have found that GRK5 binds to p53 and promotes the degradation of p53 by phosphorylation, thereby affecting p53-dependent detail. In addition, our previous studies also found that GRK5 plays an important role in the growth of tumors. In combination with previous studies, this suggests that GRK5 has many unknown new functions. The interaction proteins provide new clues for the study of new functions of GRK5.
1. The development of blood islands in GRK5 knockout mice embryos was impaired. We used the embryos of GRK5 knockout mice to observe the development of blood islands by HE staining. Compared with wild type embryos, the development of blood islands in GRK5 knockout mice embryos was impaired. Only a few embryos (about 27%) could observe normal blood islands. GRK5 is involved in the process of mouse embryo hematopoiesis.
2. Immunocoprecipitation and GST-Pull Down assays suggested that there was an interaction between GRK5 and GATA2. The interaction between GRK5 and GATA2 was found in HEK293T cells by immunocoprecipitation assay. Next, the purified GST or GST-GATA2 protein was combined with flag-HA-GRK5 and GST-coupled agarose resin through in vitro binding assay. After incubation, the immune complexes were separated by SDS-PAGE and detected by Western Blot method. It was found that there was a direct interaction between GRK5 and GATA2.
3. Peripheral blood samples were collected from the orbital venous plexus of wild-type and GRK5 knockout adult mice. Compared with wild-type mice, the number of white blood cells in the peripheral blood of GRK5 knockout adult mice was significantly reduced, mainly granulocytes, and there was no difference in the number of red blood cells and platelets.
4. By overexpressing GRK5 in human umbilical vein endothelial cells (HUVEC) and human breast cancer cells (MDA-MB-231), the proteins interacting with GRK5 were detected by immunoprecipitation/mass spectrometry. The proteins were analyzed and validated by immunoprecipitation assay. We found that these proteins can be divided into three groups: they are related to RNA shearing. Proteins; proteins associated with DNA damage repair; cytoskeleton-related proteins. These suggest that GRK5 may play a variety of previously unknown functions through these proteins interacting with GRK5
5. To sum up, we have found that GRK5 regulates the hematopoiesis of mouse embryos and its effect on the blood system of adult mice. We have also found some interacting proteins with GRK5. By immunoprecipitation/mass spectrometry, we found that some of the proteins interacting with GRK5 were involved in RNA splicing, some were associated with DNA damage and repair, and some were cytoskeleton-related proteins, which suggested that GRK5 might pass through. These proteins interacting with GRK5 play a variety of unknown functions, which may be helpful for the study of new functions of GRK5.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
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