G蛋白偶聯(lián)受體激酶5在胚胎造血過程中作用的研究及其相互作用蛋白的篩選
發(fā)布時(shí)間：2018-09-01 11:07
【摘要】：G蛋白偶聯(lián)受體(G-protein-coupled receptor, GPCR)是細(xì)胞表面最大的受體超家族,它能夠介導(dǎo)多種多樣的配體信號(hào),包括神經(jīng)遞質(zhì)、激素、氣味、光線及其它小分子,同時(shí)也介導(dǎo)多種藥物的活性,從而調(diào)節(jié)眾多的生理病理過程。G蛋白偶聯(lián)受體激酶(G-protein-coupled receptor kinases, GRKs)是調(diào)節(jié)GPCR的一類關(guān)鍵的絲/蘇氨酸激酶。當(dāng)受體被活化后,GRK通過結(jié)合并迅速磷酸化受體的胞內(nèi)段,抑制受體的持續(xù)激活,導(dǎo)致受體的內(nèi)吞和脫敏。GRK有七種亞型(GRK1-7),根據(jù)其結(jié)構(gòu)和功能的不同分為三個(gè)亞家族。其中GRK5是研究較多的成員之一,它廣泛分布于全身多種器官,尤其高表達(dá)于心臟和肺,對(duì)多種GPCR具有選擇性的調(diào)節(jié)作用,參與調(diào)節(jié)各種各樣的生理病理過程。 以往對(duì)于GRK的研究主要集中于在發(fā)育成熟的正常的機(jī)體及組織中的功能和調(diào)節(jié)作用。近年來,GRKs在胚胎發(fā)育過程中的作用也越來越受到重視,有研究發(fā)現(xiàn)：GRK2基因缺失的小鼠胚胎表現(xiàn)為生長遲緩和胚胎發(fā)育停滯,并且于胚胎期E15.5天內(nèi)死亡。我的碩士課題的第一部分工作以GRK5基因敲除小鼠為模型初步探索了GRK5在小鼠胚胎造血系統(tǒng)發(fā)育中作用及其機(jī)制；同時(shí)我們實(shí)驗(yàn)室以前的研究發(fā)現(xiàn)發(fā)現(xiàn)GRK5可以結(jié)合p53并通過使其磷酸化而促進(jìn)p53的降解,進(jìn)而影響p53依賴的細(xì)胞凋亡、細(xì)胞周期調(diào)控等重要功能；另外我們前期研究還發(fā)現(xiàn)GRK5在腫瘤的生長中起了重要的作用。也結(jié)合前人的研究,這都提示GRK5存在著許多未知的新功能。為此,我的碩士課題的第二部分工作通過免疫沉淀-蛋白質(zhì)組的方法尋找GRK5的相互作用蛋白,為研究GRK5的新的功能研究提供新的線索。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)： 1、GRK5基因敲除小鼠胚胎血島發(fā)育受損。我們采用了GRK5基因敲除小鼠的胚胎進(jìn)行切片,并通過HE染色觀察血島的發(fā)育情況,發(fā)現(xiàn)與野生型胚胎相比,GRK5基因敲除的小鼠胚胎血島發(fā)育受損,僅有較少的胚胎(約27%)可觀察到正常的血島。這提示GRK5參與小鼠胚胎造血的過程。 2、免疫共沉淀和GST-Pull Down實(shí)驗(yàn)結(jié)果提示,GRK5與GATA2之間存在著相互作用。在HEK293T細(xì)胞中通過免疫共沉淀實(shí)驗(yàn)發(fā)現(xiàn)GRK5與GATA2存在相互作用。接下來通過體外結(jié)合實(shí)驗(yàn),我們將純化的GST或GST-GATA2蛋白與Flag-HA-GRK5和GST偶聯(lián)的瓊脂糖樹脂一起孵育,得到的免疫復(fù)合物用SDS-PAGE進(jìn)行分離,再用Western Blot方法進(jìn)行檢測(cè),發(fā)現(xiàn)GRK5和GATA2存在直接的相互作用。 3、從野生型及GRK5基因敲除的成年小鼠眼眶靜脈叢采血后檢測(cè)外周血常規(guī),發(fā)現(xiàn)與野生型小鼠相比,GRK5基因敲除的成年小鼠外周血中白細(xì)胞顯著減少且主要為粒細(xì)胞的減少,紅細(xì)胞和血小板數(shù)目二者之間無差異。 4、通過分別在人臍靜脈內(nèi)皮細(xì)胞(HUVEC)和人乳腺癌細(xì)胞(MDA-MB-231)中過表達(dá)GRK5,運(yùn)用免疫沉淀/質(zhì)譜的方法檢測(cè)了與GRK5有相互作用的蛋白,分析后結(jié)合免疫沉淀實(shí)驗(yàn)也對(duì)其進(jìn)行了驗(yàn)證,我們發(fā)現(xiàn),這些蛋白可大致分為三類：與RNA剪切相關(guān)的蛋白；與DNA損傷修復(fù)相關(guān)的蛋白；細(xì)胞骨架相關(guān)蛋白。這些都提示GRK5可能通過這些與其相互作用的蛋白發(fā)揮多種多樣的目前還未知的功能 5、綜上所述,本研究發(fā)現(xiàn)了GRK5在小鼠胚胎造血中的調(diào)節(jié)作用及其對(duì)成年小鼠血液系統(tǒng)的影響,并新發(fā)現(xiàn)了一些與GRK5存在相互作用的蛋白。其中,GRK5基因敲除后會(huì)導(dǎo)致小鼠胚胎血島發(fā)育受損,且成年小鼠外周血中白細(xì)胞的數(shù)目顯著減少,而外周血中白細(xì)胞的降低主要是粒細(xì)胞絕對(duì)數(shù)的減少。通過免疫沉淀／質(zhì)譜實(shí)驗(yàn),我們發(fā)現(xiàn)與GRK5存在相互作用的蛋白中,一部分參與RNA的剪切；一部分與DNA損傷修復(fù)相關(guān)；還有一部分屬于細(xì)胞骨架相關(guān)蛋白,這些都提示GRK5可能通過這些與其相互作用的蛋白發(fā)揮多種多樣的目前還未知的功能,對(duì)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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
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