1、THE HUMEN PERSPECTIVE,Disorders Associated with G protein-Coupled Receptors,文字版,圖文版,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.,人類基因組能夠編碼多達(dá)2000中不同的G蛋白偶聯(lián)受體（GPCRs）。大約有1/4的處方藥品是作為與這類受體超家族中的某一受體的配基而發(fā)揮作用的，由此可以看出G蛋白受體的重要性。相當(dāng)一部分遺傳性紊亂都 可以上溯到GPCRs和異三聚G蛋白的缺陷。 先天性腎源性尿崩癥(CNDI)是一種罕見的遺傳性疾病，由于患病的嬰兒的腎臟缺乏產(chǎn)生終尿的能力，他們會(huì)遭受嚴(yán)

7、重的脫水。如果沒有及時(shí)地診斷治療，這種慢性的的脫水會(huì)引發(fā)智利障礙、生長遲緩、甚至是死亡。而這種紊亂的原因是腎臟的細(xì)胞失去了對后葉升壓素（抗利尿激素）的應(yīng)答能力。就像153頁提到的，一些病例的致病原因是水通道的突變，即質(zhì)膜上的水通道的改變。 然而，大多數(shù)情況是由于后葉升壓素受體出現(xiàn)錯(cuò)誤造成的。一種情況是編碼該蛋白質(zhì)的mRNA中出現(xiàn)了一個(gè)表示結(jié)束的密碼子，導(dǎo)致這個(gè)多肽的編碼提前結(jié)束了，因而使得編碼出來的產(chǎn)物變短。另一種情況是一種錯(cuò)義突變，位于第三個(gè)跨膜片段和第二個(gè)胞內(nèi)環(huán)聯(lián)結(jié)處的一個(gè)氨基酸被替代了。這樣，即使受體外表面能和后葉升壓素結(jié)合，它也不能激活G蛋白。這樣，就無法向下游的受動(dòng)器傳遞信號了。,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是由于突變導(dǎo)致被編碼受體的功能的缺失造成的，而很多導(dǎo)致信號蛋白結(jié)構(gòu)改變的突變會(huì)帶來相反的結(jié)果。這種現(xiàn)象被描述為功能的獲得。比如人們發(fā)現(xiàn)這種突變能導(dǎo)致良性的甲狀腺腫瘤，即腺瘤。我們下面來討論一下為什么會(huì)出現(xiàn)這種情況。正常的甲狀腺細(xì)胞只有在與垂體TSH（促甲狀腺

13、激素）應(yīng)答時(shí)才分泌甲狀腺素，而這些甲狀腺瘤的細(xì)胞不用與垂體激素TSH應(yīng)答就能大量分泌甲狀腺素。這些細(xì)胞中的TSH受體蛋白的一個(gè)氨基酸被替代了，從而影響了蛋白質(zhì)第三個(gè)胞內(nèi)環(huán)的結(jié)構(gòu)。這一突變使TSH受體蛋白連續(xù)不斷的刺激位于它內(nèi)表面的G蛋白，通過這一途徑連續(xù)的釋放信號，這不但會(huì)導(dǎo)致甲狀腺素的過量分泌，同時(shí)還會(huì)導(dǎo)致細(xì)胞的過量增殖從而導(dǎo)致腫瘤。這一事實(shí)已通過實(shí)驗(yàn)證實(shí)。當(dāng)向正常的沒有這種受體蛋白的培養(yǎng)細(xì)胞中轉(zhuǎn)入突變基因后，細(xì)胞顯示出有突變蛋白產(chǎn)生出來，它位于脂膜的下面，并且導(dǎo)致了cAMP在細(xì)胞內(nèi)的連續(xù)產(chǎn)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.,在病人甲狀腺正常的部分并沒有發(fā)現(xiàn)引起甲狀腺瘤的突變，這種突變只存在于病人的腫瘤組織中。這一現(xiàn)象表明，這種突變并不會(huì)遺傳，它只使甲狀腺組織中的某一個(gè)細(xì)胞增殖進(jìn)而引起腫瘤。像這樣身體內(nèi)某一個(gè)細(xì)胞突變的現(xiàn)象就叫做somatic mutation，這樣就把它與存在于全身細(xì)胞的能遺傳的突變區(qū)別開來。事實(shí)上這種somatic mutation是引起人類癌癥的首要原因。至少有一類能引起癌癥的病毒顯示出它們編碼了一類具有活性的GPCR蛋白質(zhì)。這是一種典型的皰疹病毒，它是導(dǎo)致卡波濟(jì)氏肉瘤的罪魁禍?zhǔn)住？ú?jì)氏肉瘤能在病人的皮膚上留下紫色的傷害，這種病在艾滋病患者群體中很普遍。病毒

18、的基音組編碼了白細(xì)胞殺菌素-8（interleukin-8）的受體蛋白，這種蛋白刺激了控制細(xì)胞增殖的信號途徑。,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，突變基因編碼的異三聚G蛋白亞基能夠?qū)е驴蛇z傳的紊亂現(xiàn)象。有報(bào)告，兩個(gè)男性病人患上了這樣的疾病，這些病是一些和內(nèi)分泌有關(guān)的罕見的疾病：青春期早熟和低甲狀旁腺素癥。這樣的報(bào)告可

25、以證實(shí)上面的推論。檢查發(fā)現(xiàn)，兩個(gè)病人的G構(gòu)型亞基中均有一個(gè)氨基酸被替換了，這種氨基酸序列的改變一起了突變G蛋白的兩種不同效應(yīng)。當(dāng)溫度低于正常體溫時(shí)，突變的G蛋白保持活性狀態(tài)，即使在沒有配基存在的情況下。而當(dāng)溫度為正常體溫時(shí)，無論是否有配基存在，突變的G蛋白都處于失活狀態(tài)，這一結(jié)論是在身體核心之外進(jìn)行的，溫度略低于內(nèi)臟溫度（33 vs 37）。正常情況下，當(dāng)青春期到來時(shí)，垂體LH激素分泌，內(nèi)分泌細(xì)胞對其產(chǎn)生應(yīng)答開始分泌睪丸激素。在睪丸細(xì)胞的表面上，LH與LH受體循環(huán)結(jié)合，介導(dǎo)了cAMP合成進(jìn)而雄性激素產(chǎn)生。而病人的睪丸細(xì)胞中，突變的G蛋白不需要LH配基的刺激就能產(chǎn)生cAMP，因而造成睪丸激素的提

26、前合成以及青春期的早熟。與此相反的是，存在甲狀旁腺細(xì)胞內(nèi)相同的G亞基上的突變，在溫度為37時(shí),G蛋白仍保持失活狀態(tài)。結(jié)果是甲狀旁腺細(xì)胞對于正常情況下能夠引起它們分泌甲狀旁腺素的刺激不能應(yīng)答，造成了低甲狀旁腺素癥。事實(shí)上，這些病人身體內(nèi)地的大部分器官還是以正常方式工作的，這說明對于大多數(shù)其它的細(xì)胞來講，這種特別的G構(gòu)型改變對它們的活動(dòng)沒有重要影響。,基本原理,cAMP途徑概念 細(xì)胞外信號與相應(yīng)的受體結(jié)合，導(dǎo)致細(xì)胞內(nèi)第二信使cAMP的水平變化而引起細(xì)胞反應(yīng)的信號通路。 途徑總攬 激素G蛋白耦聯(lián)受體G蛋白腺苷酸環(huán)化酶cAMP依賴cAMP的蛋白激酶A基因調(diào)控蛋白基因轉(zhuǎn)錄,首頁,cAMP信號途徑,Gs調(diào)

27、節(jié)模型 Gs與Rs相互作用，激活腺苷酸環(huán)化酶，提高cAMP水平。 Gi調(diào)節(jié)模型 Gi與Ri相互作用，抑制腺苷酸環(huán)化酶，降低cAMP水平。,G蛋白偶聯(lián)受體,右圖為G蛋白耦聯(lián)型受體該受體為7次跨膜蛋白，胞外結(jié)構(gòu)域識別胞外信號分子并與之結(jié)合，胞內(nèi)結(jié)構(gòu)域與G蛋白耦聯(lián)。通過與G蛋白耦聯(lián)，調(diào)節(jié)相關(guān)酶活性，在細(xì)胞內(nèi)產(chǎn)生第二信使，從而將胞外信號跨膜傳遞到胞內(nèi)。 圖示的八個(gè)位點(diǎn)為突變位置，這些突變會(huì)導(dǎo)致一系列的不正?，F(xiàn)象，后面詳述,24 27,G蛋白概述,三聚體GTP結(jié)合調(diào)節(jié)蛋白，簡稱G蛋白，位于質(zhì)膜內(nèi)胞漿一側(cè)，由 三個(gè)亞基構(gòu)成。二聚體通過共價(jià)結(jié)合錨于膜上起穩(wěn)定亞基的作用，而亞基本身具有GTP酶活性，能催化所結(jié)

28、合的ATP水解，恢復(fù)無活性的三聚體狀態(tài)。G蛋白在信號轉(zhuǎn)導(dǎo)過程中起著分子開關(guān)的作用。當(dāng)亞基與GDP結(jié)合時(shí)處于關(guān)閉狀態(tài)，與GTP結(jié)合時(shí)處與開啟狀態(tài)。,腺苷酸環(huán)化酶,腺苷酸環(huán)化酶是相對分子量為150KD的糖蛋白，跨膜12次。在Mg2+或Mn2+的存在下，腺苷酸環(huán)化酶催化ATP生成cAMP 。 腺苷酸環(huán)化酶是cAMP途徑中不可缺少的重要物質(zhì)，,蛋白激酶A,蛋白激酶A（Protein Kinase A，PKA）：由兩個(gè)催化亞基和兩個(gè)調(diào)節(jié)亞基組成（圖8-15），在沒有cAMP時(shí)，以鈍化復(fù)合體形式存在。cAMP與調(diào)節(jié)亞基結(jié)合，改變調(diào)節(jié)亞基構(gòu)象，使調(diào)節(jié)亞基和催化亞基解離，釋放出催化亞基。活化的蛋白激酶A催化亞

29、基可使細(xì)胞內(nèi)某些蛋白的絲氨酸或蘇氨酸殘基磷酸化，于是改變這些蛋白的活性，進(jìn)一步影響到相關(guān)基因的表達(dá),環(huán)腺苷酸磷酸二酯酶,可降解cAMP生成5-AMP，起終止信號的作用,Skip next page,Gs調(diào)節(jié)模型,返回,Gi調(diào)節(jié)模型,返回,上面已經(jīng)簡要的介紹了cAMP途徑的幾大組成部分及信號傳導(dǎo)過程。其實(shí)這個(gè)過程非常復(fù)雜，涉及到很多的環(huán)節(jié)。這么多環(huán)節(jié)當(dāng)中哪個(gè)環(huán)節(jié)出了問題，后果都是很嚴(yán)重的。比如導(dǎo)致癌變，或?qū)е驴蛇z傳性的病理改變。下面根據(jù)本文內(nèi)容，著重介紹一下G蛋白及其受體紊亂造成的疾病。,先天性腎源性尿崩癥（CNDI）,疾病屬性：罕見的遺傳性疾病,主要癥狀：患病的嬰兒的腎臟缺乏產(chǎn)生終尿的能力，他

30、們會(huì) 遭受到嚴(yán)重的脫水。如果沒有及時(shí)地診斷治療，這種慢性的的脫水會(huì)引發(fā)智障礙、生長遲緩、甚至是死亡,病理檢查：腎臟的細(xì)胞失去了對后葉升壓素（抗利尿激素）的應(yīng)答能力,為什么會(huì)出現(xiàn)這種現(xiàn)象，哪出了問題？,水通道蛋白突變導(dǎo)致腎臟重吸收能力的喪失,致病原因,圖示為腎臟的水通道蛋白(Aquaporin-2)，這是一個(gè)六次跨膜的蛋白質(zhì)，氮末端和碳末端都在膜內(nèi)，有三個(gè)細(xì)胞外環(huán)和兩個(gè)細(xì)胞內(nèi)環(huán)。圖上的黑點(diǎn)表示突變位點(diǎn)，這些突變位點(diǎn)的變化導(dǎo)致水通道蛋白的失活或通透性增強(qiáng)，相應(yīng)表現(xiàn)為病人脫水或不排尿。相應(yīng)于CNDI的突變?yōu)镼57P,致病原因,后葉升壓素受體發(fā)生錯(cuò)誤造成信號傳導(dǎo)問題導(dǎo)致CNDI,該問題分為兩種情況，一

31、種情況是編碼該蛋白質(zhì)的mRNA中出現(xiàn)了一個(gè)表示結(jié)束的密碼子，導(dǎo)致這個(gè)多肽的編碼提前結(jié)束了，因而使得編碼出來的產(chǎn)物變短。另一種情況是一種錯(cuò)義突變，位于第三個(gè)跨膜片段和第二個(gè)胞內(nèi)環(huán)聯(lián)結(jié)處的一個(gè)氨基酸被替代了。這樣，即使受體外表面能和后葉升壓素結(jié)合，它也不能激活G蛋白。這樣，就無法向下游的受動(dòng)器傳遞信號了。,上述病例中，G蛋白偶聯(lián)受體由于突變而不能和G蛋白結(jié)合，這是一種功能上的缺失。事實(shí)上來講，GCPRs的突變位點(diǎn)有很多，這些位點(diǎn)中，只有3，4兩個(gè)位點(diǎn)導(dǎo)致缺失功能的突變，剩下的位點(diǎn)均導(dǎo)致另一種突變從而使人患上另外的一些疾病，這些疾病和上述病例從癥狀上將有很大不同，但是后果更加十分嚴(yán)重。,額外功能的獲得：細(xì)胞無限增生,疾病名稱 良性甲狀腺瘤，又稱腺瘤,典型癥狀 細(xì)胞生長失控，無限增生,疾病屬性 非先天性非遺傳性疾病,額外功能的獲得：細(xì)胞無限增生,致病原因 正常的甲狀腺細(xì)胞只有在與垂體TSH（促甲狀腺激素）應(yīng)答時(shí)才分泌甲狀腺素，而這些甲狀腺瘤的細(xì)胞不用與垂體激素TSH應(yīng)答就能大量分泌甲狀腺素。這些細(xì)胞中的TSH受體蛋白的一個(gè)氨基酸被替代了，從而影響了蛋白質(zhì)第三個(gè)胞內(nèi)環(huán)的結(jié)構(gòu)。這一突變使TSH受體蛋白連續(xù)不斷的刺激位于它內(nèi)表面的G蛋白，通過這一途徑連續(xù)的釋放信號，這