G蛋白耦联受体

1、THE HUMEN PERSPECTIVE,Disorders Associated with G protein-Coupled Receptors,文字版,图文版,The human genome may encode as many as 2000 different GPCRs. Their importance in human biology is reflected by the fact that more than one-quarter of all prescription drugs act as ligands that bind to this huge super

2、family of receptors. A number of inherited disorders have been traced to defects in both GPCRs and heterotrimeric G protein . Congenital nephrogenic diabetes insipidus (CNDI) is a rare inherited disease in which infants suffer serious dehydration as the result of an inability of their kidneys to pro

3、duce a concentrated urine. If not diagnosed promptly, the chronic dehydration can produce mental retardation, inadequate growth, and even death. The disorder results from the inability of the cells of the kidneys to respond to the hormone vasopressin (antidiuretic hormone). As noted on page 153, som

4、e case of this disease result from mutations in aquaporins, The water channels of the plasma membrane.,In most case ,however, the fault lies in the vasopressin receptor, which is typically shortened as the result of a mutation that introduces a stop codon into the mRNA, causing premature termination

5、 of polypeptide synthesis (page 484). A different type of debilitating mutation in this same GPCR leads to an amino acid substitution at the junction between the third transmembrane segment and the second intracellular loop (site 4, figure 1). Even though this receptor can still bind vasopressin at

6、its external surface, the receptor cannot activate the G protein and thus cannot pass the signal downstream to the effector.,人类基因组能够编码多达2000中不同的G蛋白偶联受体（GPCRs）。大约有1/4的处方药品是作为与这类受体超家族中的某一受体的配基而发挥作用的，由此可以看出G蛋白受体的重要性。相当一部分遗传性紊乱都 可以上溯到GPCRs和异三聚G蛋白的缺陷。 先天性肾源性尿崩症(CNDI)是一种罕见的遗传性疾病，由于患病的婴儿的肾脏缺乏产生终尿的能力，他们会遭受严

7、重的脱水。如果没有及时地诊断治疗，这种慢性的的脱水会引发智利障碍、生长迟缓、甚至是死亡。而这种紊乱的原因是肾脏的细胞失去了对后叶升压素（抗利尿激素）的应答能力。就像153页提到的，一些病例的致病原因是水通道的突变，即质膜上的水通道的改变。 然而，大多数情况是由于后叶升压素受体出现错误造成的。一种情况是编码该蛋白质的mRNA中出现了一个表示结束的密码子，导致这个多肽的编码提前结束了，因而使得编码出来的产物变短。另一种情况是一种错义突变，位于第三个跨膜片段和第二个胞内环联结处的一个氨基酸被替代了。这样，即使受体外表面能和后叶升压素结合，它也不能激活G蛋白。这样，就无法向下游的受动器传递信号了。,C

8、NDI result from a mutation that leads to a loss of function of the encoded receptor. many mutation that alter the structure of signaling proteins can have an opposite effect, leading to what is described as a “gain of function.” in one such case, mutations have been found to cause a type of benign t

9、hyroid tumor, called an adenoma. Unlike normal thyroid cells that secrete thyroid hormone only in response to stimulation by the pituitary hormone TSH, the cell of these thyroid adenomas secrete large quantities of thyroid hormone without having to be stimulated by TSH (the receptor is said to act c

10、onstitutively). The TSH receptor in these cells contains an amino acid substitution that affects the structure of the third intracellular loop of the protein (figure 1,mutations at sites 5 or 6). As a result of the mutation, the TSH receptor constitutively activates a G protein on its inner surface,

11、 sending a continual signal through the pathway that leads not only to excessive thyroid hormone secretion but to the excessive cell proliferation that causes the tumor. This conclusion was verified by introducing the mutant gene into cultured cells that normally lack this receptor and demonstrating

12、 that the synthesis of the mutant protein and its incorporation into the plasma membrane led to the continuous production of cAMP in the genetically engineered cells.,CNDI是由于突变导致被编码受体的功能的缺失造成的，而很多导致信号蛋白结构改变的突变会带来相反的结果。这种现象被描述为功能的获得。比如人们发现这种突变能导致良性的甲状腺肿瘤，即腺瘤。我们下面来讨论一下为什么会出现这种情况。正常的甲状腺细胞只有在与垂体TSH（促甲状腺

13、激素）应答时才分泌甲状腺素，而这些甲状腺瘤的细胞不用与垂体激素TSH应答就能大量分泌甲状腺素。这些细胞中的TSH受体蛋白的一个氨基酸被替代了，从而影响了蛋白质第三个胞内环的结构。这一突变使TSH受体蛋白连续不断的刺激位于它内表面的G蛋白，通过这一途径连续的释放信号，这不但会导致甲状腺素的过量分泌，同时还会导致细胞的过量增殖从而导致肿瘤。这一事实已通过实验证实。当向正常的没有这种受体蛋白的培养细胞中转入突变基因后，细胞显示出有突变蛋白产生出来，它位于脂膜的下面，并且导致了cAMP在细胞内的连续产生。,The mutation that causes thyroid adenomas is not

14、 found in the normal portion of a patients thyroid but only in the tumor tissue, indicating that the mutation was not inherited but arose in one of the cells of the thyroid, which then proliferated to give rise to the tumor. A mutation in a cell of the body, such as a thyroid cell, is called a somat

15、ic mutation to distinguish it from an inherited mutation that would be present in all of the individuals cells. As will be evident in the following chapter, somatic mutations are a primary cause of human cancer. At least one cancer-causing virus has been shown to encode a protein that acts as a cons

16、titutively active GPCR. The virus is a type of herpes virus that is responsible for kaposis sarcoma, which causes purplish skin lesions and is prevalent in AIDS patients. The virus genome encodes a constitutively active receptor for interleukin-8, which stimulates signaling pathways that control cel

17、l proliferation.,在病人甲状腺正常的部分并没有发现引起甲状腺瘤的突变，这种突变只存在于病人的肿瘤组织中。这一现象表明，这种突变并不会遗传，它只使甲状腺组织中的某一个细胞增殖进而引起肿瘤。像这样身体内某一个细胞突变的现象就叫做somatic mutation，这样就把它与存在于全身细胞的能遗传的突变区别开来。事实上这种somatic mutation是引起人类癌症的首要原因。至少有一类能引起癌症的病毒显示出它们编码了一类具有活性的GPCR蛋白质。这是一种典型的疱疹病毒，它是导致卡波济氏肉瘤的罪魁祸首。卡波济氏肉瘤能在病人的皮肤上留下紫色的伤害，这种病在艾滋病患者群体中很普遍。病毒

18、的基音组编码了白细胞杀菌素-8（interleukin-8）的受体蛋白，这种蛋白刺激了控制细胞增殖的信号途径。,As noted in table 1,mutations in genes that code for the subunits of heterotrimeric G protein can also lead to inherited disorders. This is illustrated by a report on two male patients suffering from a rare combination of endocrine disorders: p

19、recocious puberty and hypoparathyroidism. Both patients were found to contain a single amino acid substitution in one of the Gisoforms. The alternation in amino acid sequence caused two effects on the mutant G protein. At temperatures below normal body temperature, the mutant G protein remained in t

20、he active state, even in the absence of a bound ligand. In contrast, at normal body temperatures, the mutant G protein was inactive, both in the presence and absence of bound ligand, the testes, which are housed outside of the bodys core, have a lower temperature than the bodys visceral organs (33 v

21、ersus 37 ). Normally, the endocrine cells of the testes initiate testosterone production at the time of puberty in response to the pituitary hormone LH, which begins to be produced at that time. The circulating LH binds to LH receptors on the surface of the testicular cells, inducing the synthesis o

22、f cAMP and subsequent production of the male sex hormone, the testicular cells of the patients bearing the G protein mutation were stimulated to synthesize cAMP in the absence of the LH ligand, leading to premature synthesis of testosterone and precocious puberty. In contrast, the mutation in this s

23、ame G subunit in the cells of the parathyroid glands,Which function at a temperature of 37, caused the G protein to remain inactive. As a result, the cells of the parathyroid gland could not respond to stimuli that would normally cause them to secrete parathyroid hormone, leading to the condition of

24、 hypoparathyroidism. The fact that most of the bodily organs functioned in a normal manner in these patients suggests that this particular G isoform is not essential in the activities of most other cells.,就像表一中提到的，突变基因编码的异三聚G蛋白亚基能够导致可遗传的紊乱现象。有报告，两个男性病人患上了这样的疾病，这些病是一些和内分泌有关的罕见的疾病：青春期早熟和低甲状旁腺素症。这样的报告可

25、以证实上面的推论。检查发现，两个病人的G构型亚基中均有一个氨基酸被替换了，这种氨基酸序列的改变一起了突变G蛋白的两种不同效应。当温度低于正常体温时，突变的G蛋白保持活性状态，即使在没有配基存在的情况下。而当温度为正常体温时，无论是否有配基存在，突变的G蛋白都处于失活状态，这一结论是在身体核心之外进行的，温度略低于内脏温度（33 vs 37）。正常情况下，当青春期到来时，垂体LH激素分泌，内分泌细胞对其产生应答开始分泌睾丸激素。在睾丸细胞的表面上，LH与LH受体循环结合，介导了cAMP合成进而雄性激素产生。而病人的睾丸细胞中，突变的G蛋白不需要LH配基的刺激就能产生cAMP，因而造成睾丸激素的提

26、前合成以及青春期的早熟。与此相反的是，存在甲状旁腺细胞内相同的G亚基上的突变，在温度为37时,G蛋白仍保持失活状态。结果是甲状旁腺细胞对于正常情况下能够引起它们分泌甲状旁腺素的刺激不能应答，造成了低甲状旁腺素症。事实上，这些病人身体内地的大部分器官还是以正常方式工作的，这说明对于大多数其它的细胞来讲，这种特别的G构型改变对它们的活动没有重要影响。,基本原理,cAMP途径概念 细胞外信号与相应的受体结合，导致细胞内第二信使cAMP的水平变化而引起细胞反应的信号通路。 途径总揽 激素G蛋白耦联受体G蛋白腺苷酸环化酶cAMP依赖cAMP的蛋白激酶A基因调控蛋白基因转录,首页,cAMP信号途径,Gs调

27、节模型 Gs与Rs相互作用，激活腺苷酸环化酶，提高cAMP水平。 Gi调节模型 Gi与Ri相互作用，抑制腺苷酸环化酶，降低cAMP水平。,G蛋白偶联受体,右图为G蛋白耦联型受体该受体为7次跨膜蛋白，胞外结构域识别胞外信号分子并与之结合，胞内结构域与G蛋白耦联。通过与G蛋白耦联，调节相关酶活性，在细胞内产生第二信使，从而将胞外信号跨膜传递到胞内。 图示的八个位点为突变位置，这些突变会导致一系列的不正常现象，后面详述,24 27,G蛋白概述,三聚体GTP结合调节蛋白，简称G蛋白，位于质膜内胞浆一侧，由 三个亚基构成。二聚体通过共价结合锚于膜上起稳定亚基的作用，而亚基本身具有GTP酶活性，能催化所结

28、合的ATP水解，恢复无活性的三聚体状态。G蛋白在信号转导过程中起着分子开关的作用。当亚基与GDP结合时处于关闭状态，与GTP结合时处与开启状态。,腺苷酸环化酶,腺苷酸环化酶是相对分子量为150KD的糖蛋白，跨膜12次。在Mg2+或Mn2+的存在下，腺苷酸环化酶催化ATP生成cAMP 。 腺苷酸环化酶是cAMP途径中不可缺少的重要物质，,蛋白激酶A,蛋白激酶A（Protein Kinase A，PKA）：由两个催化亚基和两个调节亚基组成（图8-15），在没有cAMP时，以钝化复合体形式存在。cAMP与调节亚基结合，改变调节亚基构象，使调节亚基和催化亚基解离，释放出催化亚基。活化的蛋白激酶A催化亚

29、基可使细胞内某些蛋白的丝氨酸或苏氨酸残基磷酸化，于是改变这些蛋白的活性，进一步影响到相关基因的表达,环腺苷酸磷酸二酯酶,可降解cAMP生成5-AMP，起终止信号的作用,Skip next page,Gs调节模型,返回,Gi调节模型,返回,上面已经简要的介绍了cAMP途径的几大组成部分及信号传导过程。其实这个过程非常复杂，涉及到很多的环节。这么多环节当中哪个环节出了问题，后果都是很严重的。比如导致癌变，或导致可遗传性的病理改变。下面根据本文内容，着重介绍一下G蛋白及其受体紊乱造成的疾病。,先天性肾源性尿崩症（CNDI）,疾病属性：罕见的遗传性疾病,主要症状：患病的婴儿的肾脏缺乏产生终尿的能力，他

30、们会 遭受到严重的脱水。如果没有及时地诊断治疗，这种慢性的的脱水会引发智障碍、生长迟缓、甚至是死亡,病理检查：肾脏的细胞失去了对后叶升压素（抗利尿激素）的应答能力,为什么会出现这种现象，哪出了问题？,水通道蛋白突变导致肾脏重吸收能力的丧失,致病原因,图示为肾脏的水通道蛋白(Aquaporin-2)，这是一个六次跨膜的蛋白质，氮末端和碳末端都在膜内，有三个细胞外环和两个细胞内环。图上的黑点表示突变位点，这些突变位点的变化导致水通道蛋白的失活或通透性增强，相应表现为病人脱水或不排尿。相应于CNDI的突变为Q57P,致病原因,后叶升压素受体发生错误造成信号传导问题导致CNDI,该问题分为两种情况，一

31、种情况是编码该蛋白质的mRNA中出现了一个表示结束的密码子，导致这个多肽的编码提前结束了，因而使得编码出来的产物变短。另一种情况是一种错义突变，位于第三个跨膜片段和第二个胞内环联结处的一个氨基酸被替代了。这样，即使受体外表面能和后叶升压素结合，它也不能激活G蛋白。这样，就无法向下游的受动器传递信号了。,上述病例中，G蛋白偶联受体由于突变而不能和G蛋白结合，这是一种功能上的缺失。事实上来讲，GCPRs的突变位点有很多，这些位点中，只有3，4两个位点导致缺失功能的突变，剩下的位点均导致另一种突变从而使人患上另外的一些疾病，这些疾病和上述病例从症状上将有很大不同，但是后果更加十分严重。,额外功能的获得：细胞无限增生,疾病名称 良性甲状腺瘤，又称腺瘤,典型症状 细胞生长失控，无限增生,疾病属性 非先天性非遗传性疾病,额外功能的获得：细胞无限增生,致病原因 正常的甲状腺细胞只有在与垂体TSH（促甲状腺激素）应答时才分泌甲状腺素，而这些甲状腺瘤的细胞不用与垂体激素TSH应答就能大量分泌甲状腺素。这些细胞中的TSH受体蛋白的一个氨基酸被替代了，从而影响了蛋白质第三个胞内环的结构。这一突变使TSH受体蛋白连续不断的刺激位于它内表面的G蛋白，通过这一途径连续的释放信号，这