CST-2-GPCR and downstream signal-new

1、GTP- Binding Proteins and Signal TransductionG-protein coupled receptorG-proteinEffectors downstream GPCROther GTP-binding proteinGPCR superfamily are also referred to as seven-transmembrane (7TM).These receptors translate the binding of extracellular signaling molecules into the activation of GTP-b

2、inding proteins.Science 23 Nov. 2007: Vol. 318. no. 5854, p.1258-1265.2.1 G ProteinCoupled Receptors2.1 G ProteinCoupled ReceptorsGPCRs constitute the largest gene family in the human genome (950 genes), 500 of which are sensory.They are the target of the majority of best-selling drugs (40%-50% of a

3、ll prescription pharmaceuticals on the market). 高血压药物Cozaar(洛沙坦，血管紧张素II抑制剂) 乳腺癌药物Zoladex(诺雷得，醋酸戈舍瑞林，促黄体生成素释放激素的一种类似物) 焦虑药物Buspar、精神分裂药物Clozaril Zantac（善胃得，组胺H2-受体拮抗剂） Claritin（开瑞坦，氯雷他定）2000亿美元2.1.1 Structure of GPCRYellow: hydrophobic residuesGrey: non-polar residuesBlue: polar residuesRed: targets

4、for GRK2Black: targets for PKA GPCRs contain seven transmembrane helices, but the 7TM receptors have few conserved residues. Many (if not most) 7TM receptors function not as monomers, but as dimers or oligomeric clusters. 2.1.2 GPCR Function1.The visual sense: the opsins (视蛋白) use a photoisomerizati

5、on (异构化) reaction to translate electromagnetic radiation into cellular signals. Rhodopsin (视紫红质) , for example, uses the conversion of 11-cis-retinal (视网膜) to all-trans-retinal for this purpose.2.The sense of smell: receptors of the olfactory (嗅觉) epithelium bind odorants (olfactory receptors) and p

6、heromones (信息素) (vomeronasal (鼻骨的) receptors).3.Behavioral and mood regulation: receptors in the mammalian brain bind several different neurotransmitters, including serotonin and dopamine.4.Regulation of immune system activity and inflammation: chemokine receptors bind ligands that mediate intercell

7、ular communication between cells of the immune system; receptors such as histamine receptors bind inflammatory mediators and engage target cell types in the inflammatory response.5.Autonomic nervous system transmission: both the sympathetic (交感的) and parasympathetic (副交感的) nervous systems are regula

8、ted by GPCR pathways. These systems are responsible for control of many automatic functions of the body such as blood pressure, heart rate and digestive processes.2.1.3 GPCR- classificationClass A: Rhodopsin (视紫红质) family Class B: Secretin familyClass C: Metabotropic glutamate and GABA familyFrizzle

9、d class: Adhesion family：2.1.3 GPCR- classification* Class A Rhodopsin family o Amine o Peptide o Hormone protein o (Rhod)opsin o Olfactory o Prostanoid (前列腺素类) o Nucleotide-like o Cannabinoid (大麻) o Platelet activating factor o Gonadotropin(促性腺激素)-releasing hormone o Thyrotropin-releasing hormone o

10、 Melatonin (褪黑素) o Leukotriene(白三烯) B4 receptor* Class B Secretin family o Calcitonin (降钙素) o Corticotropin releasing factor (促皮质释放因子) o Gastric inhibitory peptide o Glucagon (胰高血糖素) o Growth hormone-releasing hormone o Parathyroid hormone o Secretin (分泌素) o Vasoactive(血管活性) intestinal polypeptide o

11、 Diuretic hormone (利尿素) o Brain-specific angiogenesis inhibitor (BAI) o Cadherin(钙粘素)2.1.4 GPCR-ligand binding普萘洛尔(心得安) -受体阻断剂propranolol Binding of low molecular mass ligands2.1.4 GPCR-ligand bindingBinding of peptide or protein ligands2.1.4 GPCR-ligand binding2.2 G proteinsG proteinsGTPasesGTP-bin

12、ding Proteins2.2 G proteinsG proteinsThe class of GTP-binding proteins that interact with 7-transmembrane receptors.Heterotrimeric G-proteins were discovered and characterized by Martine Rodbell and his colleagues at the National Institutes in the early 1970s. These proteins are referred to as G pro

13、teins because they bind guanine nucleotides, either GDP or GTP Heterotrimeric: a-subunit and bg-subunita-subunit contains the nucleotide binding siteAnchored to the membrane by lipid attachments, one at the N-terminal of the a-subunit and the other at the C-terminal of the g-subunit. Who binds GDP/G

14、TPWhere do they locate2.2.1 Trimeric G Proteins Relay Signals from GPCRs2.2.1 Trimeric G Proteins Relay Signals from GPCRs When an extracellular signal molecule binds to a GPCR, the receptor undergoes a conformational change that enables it to activate a trimeric GTP-binding protein (G protein). a-s

15、ubunit contains the nucleotide binding site Anchored to the membrane by lipid attachments, one at the N-terminal of the a-subunit and the other at the C-terminal of the g-subunit. 2.2.2 G proteins-ActivationG protein remains associated with the effector enzyme.Upon stimulation and association of the

16、 receptor with the G protein/effector enzyme complex, the GDP dissociates, replaced by GTP.Following activation, the contact between the G protein and the agonist-receptor complex is weakened, allowing the receptor to detach and seek out further inactive G protein molecules.After the hydrolysis of G

17、TP to GDP, the G protein/effector enzyme complex return to their inactive forms2.3 a subunitsThe Ras-like domain possess a nucleotide binding site and binding surfaces for bg-subunits.The sis-helix bundle domain contribute to nucleotide binding and assist the hydrolysis of GTP.The sis-helix bundle d

18、omain acts as an allosteric modulator, enhancing the efficiency of the interaction of the activated a subunit with its effectors.2.3.1 Discovery of a subunits异丙肾上腺素LymphomaCell line S49Elevation of cAMP has the effect of arresting the cell cycle in the G1 phase and then promoting cell death.Forskoli

19、n, a direct activator of adenylyl cyclase2.3.2 Classification of a subunitsa subunits determine G protein diversity.There are only 16 genes specifying the a subunits in animals and provided about 20 gene products. Why the number of products is more than that of the genes? Most animal cells express 9

20、 or 10 a subunits. Some such as at, aolf are expressed only in single classes of sensory cells.Brutons tyrosine kinaseGPCR- classification according to a subunit2.3.2 Classification of a subunits2.3.3 a a subunits interact with othersThe C-terminal region dictates the specificity of interaction with

21、 receptors.The N-terminal glycines of Gi and Go are the sites of attachment of myristic acid(肉蔻酸), a modification that facilitates heterotrimer formation.Conformational changes when GDP is exchanged for GTP.Core region of aiSWI: white to yellowSWII: pink to redSWIII: light blue to dark blueDetach of

22、 bg-subunits from a subunits 2.4 bg subunitsThere are 5 b subtypes and at least 12 g-subtypes. But not all kinds of the potential bg pairs exist in nature.The association between the a-subunit with the bg-subunits is not definitely specific but sometime yes for some special receptors. 2.4 bg subunit

23、sMammalian g-subunits are post-translationally modified at the C-terminus by the addition of the 20-carbon geranylgeranyl group (4 isoprene units, 四个异戊二烯单元) or the 15-carbon farnesyl group (法尼酰基), which tethered to the membrane.2.4 bg subunitsbgbg subunits are also signaling proteins. They ensure th

24、e localization, effective coupling and deactivation of the a-subunits. They regulate the affinity of the receptors for their activating ligands.They reduce the tendency of GDP to dissociate from a-subunits and thus stabilizing the inactive state.They interact directly with some downstream effector s

25、ystems.Cardiac K+ channelPhosphoinositide 3-kinase g b- Phospholipase CThey regulate receptor phosphorylation by specific receptor kinases.Activation of GPCR2.5 Effectors downstream GPCR Overview of GPCR pathway The effector enzymes coupled to GTP binding proteins include adenylyl cyclase and phosph

26、olipase C, which produce the second messengers（cAMP, IP3 & DAG）. PKA, PKC, PLC, PI3K, are involved downstream GPCR. 腺苷酸环化酶腺苷酸环化酶-cAMP-PKA激活激活AC抑制抑制AC 磷脂酶磷脂酶CIP3-Ca2+DAG-PKC 离子通道离子通道 MAPK、PI3K信号通路信号通路2.5 Effectors downstream GPCR2.5.1 G Proteins Regulate the Production of Cyclic AMPCyclic AMP (cA

27、MP) cAMP acts as a small intracellular mediator in all kinds of cells. Its normal concentration in the cytosol is about 107 M, but an extracellular signal can increase this concentration more than twentyfold in seconds. Cyclic AMP is synthesized from ATP by a plasma-membrane-bound enzyme adenylyl cy

28、clase (AC), and its rapidly and continuously destroyed by cAMP phosphodiesterases that hydrolyze cAMP to 5-AMP.This nerve cell in culture is responding to the neurotransmitter serotonin (5羟色胺), which acts through a GPCR to cause a rapid rise in the intracellular concentration of cAMP.Adenylyl cyclas

29、e is a large multipass transmembrane protein with its catalytic domain on the cytosolic side of the plasma membrane. There are at least eight isoforms in mammals, most of which are regulated by both G proteins and Ca2+. GPCRs that coupled to a stimulatory G protein (Gs) can increase cAMP concentrati

30、on by activates adenylyl cyclase. Another G protein, called inhibitory G protein (Gi), inhibits adenylyl cyclase.Adenylyl cyclase-regulationCholera toxin 霍乱毒素cAMP-dependent Activation of PKA In most animal cells, cAMP exerts its effects mainly by activating cAMP-dependent protein kinase (PKA). PKA p

31、hosphorylates specific serines or threonines on selected target proteins PKA consists of two catalytic subunits and two regulatory subunits. cAMP binds to the regulatory subunits and alters their conformation, causing them to dissociate from the complex. The released catalytic subunits are thereby a

32、ctivated.Regulation of gene expression by cAMP-PKA Some responses mediated by cAMP depend on changes in the transcription of specific genes and take hours to develop fully. The cAMP response element (CRE) is found in the regulatory region of many genes activated by cAMP. A specific gene regulatory p

33、rotein called CRE-binding protein (CREB) recognizes CRE sequence. When PKA is activated by cAMP, it phos-phorylates CREB on a single serine; phosphorylated CREB then recruits a transcriptional coactivator called CREB-binding protein (CBP), which stimulates the transcription of the target genes.PKA M

34、ediates Most of the Effects of cAMP Over one hundred PKA substrates have been described. Most of these carry out different functions. How PKA phosphorylates the appropriate substrates in response to a particular stimulus, in a particular cell type?2.5.2 GPCR Mediated Activation of Phospholipase C Ma

35、ny GPCRs exert their effects mainly via G proteins called Gq that activate the plasma membrane-bound enzyme phospholipase C-b (PLCb). The activated PLCb then cleaves the PIP2 to generate two products: inositol 1,4,5-trisphosphate (IP3) diacylglycerol (DAG). IP3 and Ca2+Inositol 1,4,5-Trisphosphate (

36、IP3) Triggers Release of Ca2+ from the Endoplasmic ReticulumCa2+ Functions as a Ubiquitous Intracellular Mediator The most important Ca2+-binding proteins is calmodulin (钙调蛋白) which help to relay the cytosolic Ca2+ signal, Calmodulin functions as a multipurpose intracellular Ca2+ receptor, governing

37、 many Ca2+-regulated processes. It consists of a highly conserved, single polypeptide chain with four high-affinity Ca2+-binding sites. When activated by Ca2+ binding, it undergoes a conformational change, which enables calmodulin to bind to various target proteins in the cell to alter their activit

38、y. An activated calmodulin binds to its target protein and further changes its conformation. Calmodulin regulates many targets including enzymes and membrane transport proteins. As one example, Ca2+/calmodulin binds to and activates the plasma membrane Ca2+-pump that uses ATP hydrolysis to pump Ca2+

39、 out of cells.Ca2+ Functions as a Ubiquitous Intracellular Mediator Many effects of Ca2+, however, are mediated by a family of serine/threonine protein kinases called Ca2+/calmodulin-dependent kinases (CaM-kinases). Some CaM-kinases phosphorylate gene regulatory proteins, such as the CREB protein, a

40、nd in this way activate or inhibit the transcription of specific genes. One of the best-studied CaM-kinases is CaM-kinase II, which is found in most animal cells but is especially enriched in the nervous system. It constitutes up to 2% of the total protein mass in some regions of the brain, and it i

41、s highly concentrated in synapses. It seems to have a role in some types of memory and learning in the vertebrate nervous system. Mutant mice that lack a brain-specific form of the enzyme have specific defects in their ability to remember where things are.Ca2+ Functions as a Ubiquitous Intracellular

42、 Mediator2.5.3 Ion Channels Regulated by GPCRG proteins directly activate or inactivate ion channels in the plasma membrane of the target cell, thereby altering the ion permeability and hence the electrical excitability of the membrane.2.5.1 Muscarinic acetylcholine receptors and K+ channels in myoc

43、ardial cell Acetylcholine reduces both the rate and strength of heart muscle cell contraction, which is mediated by a muscarinic acetylcholine receptors that activate the Gi protein. Once activated, the a subunit of Gi inhibits adenylyl cyclase, while the bg subunits bind to K+ channels in the heart

44、 muscle cell plasma membrane and open them. The opening of these K+ channels makes it harder to depolarize the cell and thereby contributes to the inhibitory effect of acetylcholine on the heart.GPCRs Regulate Cyclic-Nucleotide-Gated Ion Channels Smell：Humans can distinguish more than 10,000 distinc

45、t smells. Olfactory receptor neurons use specific GPCRs called olfactory receptors to recognize odors; the receptors are displayed on the surface of the neurons cilia. The receptors act through cAMP. When stimulated by odorant binding, they activate an olfactory-specific G protein (known as Golf), w

46、hich in turn activates adenylyl cyclase. The resulting increase in cAMP opens cAMP-gated cation channels, thereby allowing an influx of Na+, which depolarizes the olfactory receptor neuron and initiates a nerve impulse that travels along its axon to the brain.Vision Depend on GPCRs That Regulate cGMP-Gated Ion Channels In visual transduction responses are mediated by GPCR that coupled Gt-protein, which has been well studied in rod photoreceptors (rods) in the vertebrate retina. Rhodopsin (视紫红质) is t