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Chapter 14 Signal-Transduction Pathways G proteins Signal-transduction circuits in biological systems have molecular on-off switches that, like those in a computer chip, transmit information when “on”. Common among these circuits are those including G proteins, which transmit a signal when bound to GTP and are silent when bound to GDP Outline 14.1 Heterotrimeric G proteins transmit signals and reset themselves 14.2 Insulin signaling: Phosphorylation cascades are central to many signal-transduction processes 14.3 EGF signaling: signal-transduction systems are poised to respond 14.4 Many elements recur with variation in different signal-transduction pathways 14.5 Defects in signal-transduction pathways can lead to cancer and other diseases 2 Signal Transduction Pathways • Signal Transduction – chain of events that converts the message “this molecule is present” to a physiological response β-cells in pancreas Adrenal glands Epinephrine腎上腺素 Epinephrine + β-Adrenergic receptor Energy-store mobilization insulin胰島素 insulin + Insulin receptor Epidermal growth factor (EGF) + EGF receptor Increased glucose Uptake/ glycogen storage Expression of growth promoting gene (wound repair) Fig 14.1 Three signal –transduction pathways. The binding of signaling molecules to their receptors initiates pathways that lead to important physiological 3 response Key Steps in Signal Transduction •Release of the primary messenger Hormone •Reception of the primary messenger (ligand) Feedback pathways •Delivery of the message inside the cell by the second messenger Reception cell-surface receptor •Activation of effectors that directly alter the physiological response Amplification •Termination of the Signal Signal Transduction Response(s) Fig 14.2 Principles of signal transduction 4 Common Second Messengers •Second messengers are intracellular molecules – relay information from the receptor-ligand complex – change in concentration in response to environmental signals Cyclic AMP – Mediate the next step in the molecular information circuit •Signal may be amplified •Often free to diffuse throughout cell •Common second messengers may create “cross talk” Fig 14.3 common second messenger 5 14.1 Heterotrimeric G Proteins Transmit Signals and Reset Themselves •Epinephrine –A hormone secreted by the adrenal glands of mammals in response to internal and external stressor –Signaling begins with ligand (Epinephrine) bind to β-adrenergic receptor (β-AR) •A member of seven-transmembrane-helix (7TM) receptors >20,000 receptors serpentine (蜿蜒的,蛇的) receptors: the single polypeptide chain "snakes" Fig 14.4 The 7TM receptor through the membrane seven times 6 14.1 Heterotrimeric G Proteins Transmit Signals and Reset Themselves •Rhodopsin 視紫質(Chapter 33) –A well-characterized member of the 7TM receptor family –retina (視網膜) protein •senses the presence of photons (ligand) •initiate the signaling cascade responsible for visual sensation –lysine residue within rhodopsin is covalently modified by a form of vitamin A 11-cis-retinal •exposure to light induces the isomerization of 11-cisretinal to its all trans form, producing structural changes in the receptor Fig 14.5 (A) Structures of •initiation of an action potential as visual stimulusrhodopsin 7 •β2-adrenergic receptor –Identified the structure by the inhibitor, carazolol, competes with epinephrine –Similarities with rhodopsin –Mechanism •Binding of a ligand from outside the cell induces a structural rearrangement in the part of the 7TM receptor that is positioned inside the cell Fig 14.5 (B) Structures of β2adrenergic receptor 8 Ligand Binding to 7TM Receptors Leads to the Activation of heterotrimeric G Proteins G proteins, also known as guanine •Epinephrine bind to β2nucleotide-binding proteins adrenergic receptor conformational change of cytoplasmic domain activated G protein activate adenylate cyclase catalyzed the conversion of ATP into cyclic AMP (cAMP) activate Protein kinase A Fig 14.6 Activation of protein kinase A by9a G-protein pathway G Proteins Cycle Between GDP- and GTPBound Forms •G protein –Unactivated state: G protein is bound to GDP •Heterotrimer consisting of α, β, and γ subunits – α Subunit (Gα) binds the nucleotide » A member of the P-loop NTPase family » Participate in nucleotide binding – α and γ subunit anchor to the membrane Fig 14.7 A heterrtrimeric G protein –Hormone-bound receptor is catalyze the exchange of GTP for bound GDP •GTP binding induces conformational change •Decreases Gα affinity for Gβγ Gα dissociation • Gα can now bind to other proteins (adenylate cyclase) Gαs (s =stimulatory) Because signal through G protein 7TM receptors are called G-protein coupled receptors (GPCRs) 10 •Gα binding to adenylate cyclase –a membrane protein that contains 12 membrane-spanning helices •two large cytoplasmic domains form the catalytic part of the enzyme –Converts ATP into cAMP Fig 14.8 Adenylate cyclase activation Catalytic fragment 11 What does the cAMP do? •Increase concentration of cAMP can effect a wild range of cellular processes: –cAMP stimulates production of ATP for muscle contraction –Enhance the degradation of fuel stores –Increase the secretion of acid by gastric mucosa –Leads to the dispersion of melanin pigment granules –Diminishes the aggregation of blood platelets –Induces the opening of chloride channels Most effect of cAMP in eukaryotic cells are mediated by the activation of a single protein kinase: protein kinase A (PKA) 12 •Protein kinase A –Consists two regulatory (R) chain and two catalytic (C) chain (Ch10) •In the absence of cAMP inactive •cAMP binds to the R chains releases the catalytic chains active –Activated PKA then phosphorylates specific serine and threonine in many targets to alter their activity • PKA phosphorylates two enzymes that lead to the breakdown of glycogen (Ch21) •PKA stimulates the expression of specific genes by phosphorylating a transcriptional activator called cAMP response element binding (CREB) protein gene expression 13 Epinephrine signaling pathway –On binding of ligand, the receptor activates a G protein that in turn activates the enzyme adenylate cyclase. –Adenylate cyclase generates the second messenger cAMP. – The increase in cAMP results in a biochemical response to the initial signal Fig 14.9 Epinephrine signaling pathway 14 • How is the signal initiated by epinephrine switched off? 1. G proteins spontaneously reset themselves through GTP Hydrolysis • Intrinsic GTPase activity of Gα -- hydrolyze bound GTP to GDP and Pi. – the bound GTP acts as a built-in clock that spontaneously resets the Gα subunit after a short time period. – After GTP hydrolysis and release the Pi, the GDP-bound form of Gα then reassociates with Gβγ to re-form the inactive heterotrimeric protein. Fig 14.10 Reseting Gα. Intrinsic GTPase activity 15 heterotrimeric protein • How is the signal initiated by epinephrine switched off? 1. G proteins spontaneously reset themselves through GTP Hydrolysis 2. Signal termination 1. Hormone dissociates, returning the receptor to its initial, unactivated state 2. the hormone-receptor complex activates a kinase that phosphorylates serine and threonine residues in the carboxylterminal tail of the receptor. – Diminishes the ability to activate G protein β-adrenergic receptor kinase Fig 14.11 Signal termination. (G-protein receptor kinase 2, GRK2) 16 Some 7TM receptors activate the phosphoinositide cascade •phosphoinositide cascade –angiotensin II (血管緊縮素) (ligand, peptide hormone, control blood pressure) -- angiotensin II receptor –angiotensin II receptor activates Gαq protein –GTP-form Gαq binds to activate the β isoform of phospholipase C •Catalyzes the cleavage of PIP2 into inositol 1,4,5-triphosphate (IP3) and Diacylglycerol (DAG) which stays in the membrane Fig 14.12 phospholipase C reaction. 17 •inositol 1,4,5-triphosphate (IP3) – soluble and diffuses away from the membrane – causes the rapid release of Ca2+ from intracellular stores in the endoplasmic reticulum •specific IP3 -gated Ca2+ -channel proteins in the ER membrane open to allow calcium ions to flow from the ER into the cytoplasm – Elevated level of cytoplasmic Ca2+ triggers smooth muscle contraction, glycogen breakdown, and vesicle release Calcium is also a signal molecule: it can bind proteins called calmodulin and enzymes such as protein kinase C Fig 14.13 phosphoinositide cascade 18 . •Diacylglycerol (DAG) –remains in the plasma membrane – activates protein kinase C (PKC) •phosphorylates serine and threonine residues in many target proteins. •the specialized DAG binding domains of this kinase require bound calcium. IP3 increases the Ca2+ concentration, and Ca2+ facilitates the DAG-mediated activation of protein kinase C. Fig 14.13 phosphoinositide cascade Both IP3 and DAG act transiently because they are . converted into other species by phosphorylation or other processes. 19 Ca2+ is a widely used second messenger •Ca2+ participates in many signaling processes, the properties: 1. Fleeting changes in [Ca2+] are readily detected •intracellular levels are low to prevent the precipitation of carboxylated and phosphorylated compounds 2. can bind tightly to proteins and induce substantial structural rearrangements •bind well to negatively charged oxygen atoms (side chains of glutamate and aspartate) and uncharged oxygen atoms (main-chain carbonyl groups and sidechain oxygen atoms from glutamine and asparagine). •Ca2+ can coordinated multiple ligands -from six to eight oxygen atoms- crosslink different protein segments and induce significant conformational changes Fig 14.14 calcium-binding site. 20 •Scientists can monitor cellular [Ca2+] in real –Fura-2 (molecular-imaging agents) can bind Ca2+ and change their fluorescent properties on Ca2+ binding •Fura-2 binds Ca2+ through appropriately positioned oxygen atoms (in red) within its structure •When Fura-2 introduced into cells, change in available Ca2+ by detection changes in fluorescence Fig 14.15 calcium imaging Red: high Ca2+ 21 Calcium ion often activates the regulatory protein calmodulin •Calmodulin (CaM) –a 17-kd protein with four Ca2+-binding sites •Member of EF-hand protein family – EF-hand is a Ca2+ binding motif: Helix, loop, helix motif Fig 14.16 EF hands 2+ – 7 seven oxygen are coordinated to each Ca –serves as a calcium sensor in nearly all eukaryotic cells –At cytoplasmic concentrations above about 500 nM, Ca2+ binds to and activates calmodulin •Conformational changes expose hydrophobic residues •Newly exposed surfaces can bind other proteins, eg, CaM kinase I, and stimulating them phosphorylate proteins Calmodulin-dependent protein kinase 22 Fig 14.17 calmodulin binds to the α helices. •Signal transduction pathway: –Secondary messenger is increased : Ca2+ –The signal is sensed by a second-messenger-binding protein: calmodulin –Second-messenger-binding protein acts to generate changes in enzyme : calmodulin-dependent kinases •Phosphorylate many different proteins – Regulate fuel metabolism (調節代謝) – Ionic permeability (改變離子通透) – Neurotransmitter synthesis (神經傳導物質的合成) – Neurotransmitter release (神經傳導物質的釋放) 23 14.2 Insulin signaling: Phosphorylation cascades are central to many signaltransduction processes •Signal transduction pathways initiated by receptors kinases as part of the receptor structures Insulin signaling Focus on “ leads to the mobilization of glucose transporters to the cell surface” •Insulin –The hormone released in response to increased blood- glucose levels –a peptide hormone that consists of two chains, linked by three disulfide bonds Fig 14.18 insulin structure 24 Insulin signaling •Insulin receptor Receptor tyrosine kinase –Dimer of two identical subunits (homodimer) •Each unit consists of one chain and one chain linked to one another by disulfide bond –Two α subunits move together around a insulin Fig 14.19 The insulin –Each β subunit has a kinase domain similar to PKA receptor – Differs from PKA (protein kinase A) • Is a tyrosine kinase •The kinase is in an inactive conformation when the domain is not covalently modified – Requires tyr in activation loop be phosphorylated 25 Insulin binding results in the cross-phosphorylation and activation of the insulin receptor •How is the activation loop phosphorylated? –the two α subunits move together to surround one insulin molecule, the kinase domains also draw closer together –the two β subunits forced together, the kinase domain catalyze the phosphoryl groups from ATP to tyrosine residues in the activation loops -- conformational change takes place – active conformation Fig 14.20 Activation of the insulin receptor by phosphorylation 26 Activated insulin-receptor kinase initiates a kinase cascade •Insulin-receptor kinase phosphorylation sites act as docking sites for other substrates, including insulinreceptor substrates (IRS) –IRS-1 and IRS-2 are two homologous proteins with a common modular structure •pleckstrin homology domain: binds phosphoinositide lipids •phosphotyrosine-binding domain anchoring the IRS protein to the insulin receptor and the membrane • Tyr-X-X-Met (YXXM): four sequences are phosphorylated by the insulin-receptor tyrosine kinase Fig 14.22 the modular structure of insulinreceptor substrates IRS-1 and IRS-2 27 IRS act as adaptor protein!! (not enzyme but serve to tether the downstream components of this signaling pathway to the membrane, eg., phosphoinositide 3-kinase) Fig 14.21 insulin signaling Fig 14.25 insulin signaling pathway 28 •lipid kinases have Src homology 2 (SH2) domains –Src homology 2 (SH2) domains bind to specific phosphotyrosine domains in the IRS protein –Lipid kinases (also called phosphoinositide 3-kinase; PI3Ks ) function on phosphorylate PIP2 to PIP3 Fig 14.23 Structure of the SH2 domain Fig 14.24 Action of a lipid kinase in insulin signaling 29 •PIP3 activates a protein kinase PDK1 (with a pleckstrin homology domain that specific for PIP3) •PDK1 activates another protein kinase Akt –Akt not membrane anchored, and moves through the cell to phosphorylates targets •control the trafficking of the glucose receptor GLUT4 to the cell surface •target enzymes that stimulate glycogen synthesis 30 Insulin signaling pathway 31 Effect of insulin on glucose uptake and metabolism •Insulin binds to its receptor, which in turn starts many protein activation cascades, include: – translocation of Glut-4 transporter to the plasma membrane and influx of glucose –Glycogen synthesis –Glycolysis –fatty acid synthesis 32 http://en.wikipedia.org/wiki/GLUT4 Insulin signaling is terminated by the action of phosphatases •In insulin signaling, three classes of enzymes are of particular importance in shutting off the signaling pathway: –Protein tyrosine phosphatases: remove phosphoryl groups from tyrosine residues on the insulin receptor and the IRS adaptor proteins –Lipid phosphatases: hydrolyze PIP3 to PIP2 –Protein serine phosphatases: remove phosphoryl groups from activated protein kinases such as Akt 33 14.3 EGF signaling: signal-transduction systems are poised to respond •EGF Signaling –Signal molecule epidermal growth factor (EGF) binds to a receptor tyrosine kinase (EGF receptor; EGFR) that participates in cross-phosphorylation reactions •Epidermal growth factor (EGF) –a 6-kd polypeptide that stimulates the growth of epidermal and epithelial cells Fig 14.26 Structure of epidermal growth factor 34 •EGF receptor –A dimer of two identical subunit •Exits as monomers until they bind EGF –EGF-binding domain that lies outside the cell •Dimerization is mediated by a dimerization arm •the dimer binds two ligand molecules –a single transmembrane helix-forming region –the intracellular tyrosine kinase domain •participates in cross-phosphorylation reactions – One unit phosphorylated by another unit within a dimer •Not within the activation loop of kinase –the tyrosine-rich domain at the carboxyl terminus •5 tyrosines are phosphorylated Binding of EGF to the extracellular domain causes the receptor to dimerize and undergo cross-phosphorylation and activation Fig 14.27 Molecular structure of EGF receptor 35 Fig 14.28 EGF receptor dimerization •Why doesn’t the receptor dimerized and signal in the absence of EGF? –Conformational different •The dimerization arm binds to a domain within the same monomer •Hold the receptor in a closed configuration •makes it unavailable for interaction with the other receptor Fig 14.29 Structure of the unactivated EGF receptor 36 EGF signaling leads to the activation of Ras, a small G protein •After EGF receptor phosphorylation –The SH2 domain of an adaptor protein, Grb-2, binds to the phosphotyrosine residues of the EGF receptor –Grb-2 binds Sos using two Src homology 3 (SH3) domains • SH3 domains bind proline-rich polypeptides – Sos, in turn, binds to Ras and activates it • Ras in a class of proteins called “small G proteins” – GDP-Ras GTP-Ras •Sos as a guanine-nucleotide-exchange factor (GEF) Structure of Grb-2, an Adaptor Protein Fig 14.30 Ras activation 37 mechanism Activated Ras initiates a protein kinase cascade •GDP-Ras GTP-Ras change conformation –Binds other proteins, including Raf, a protein kinase (protein-protein interaction) –Raf then undergoes a conformational change that activates its kinase domain •Ras and Raf are anchored to membrane, through a covalently bound isoprene lipid –Raf phosphorylates other proteins, including the kinases termed MEKs –MEKs activate “extracellular signal-regulated kinases” (ERKs) –ERKS phosphorylate many substrates, including other kinases and transcription factors 38 Ras and Raf Signal Transduction (or other extracellular signaling molecule) Protein tyrosine kinase domain Dimer activated adapter phosphorylation MAPK/ERK kinase phosphorylation phosphorylation Extracellular-signalregulated kinase phosphorylation Fig 12.36 Enhanced transcription 12-39 More cell division EGF signaling pathway Fig 14.31 EGF signaling pathway EGF signaling is terminated by protein phosphatases and the intrinsic GTPase activity of Ras •protein phosphatases play key roles in the termination of EGF signaling •Signal activation also initiates signal termination –Ras possesses intrinsic GTPase activity –The activated GTP form of Ras spontaneously converts into the inactive GDP form –GTPase-activating proteins (GAPs) interact with small G proteins in the GTP-bound form and facilitate GTP hydrolysis 41 Small G proteins or small GTPases •Difference between small G proteins and heterotrimeric G proteins Size small G proteins heterotrimeric G proteins 20-25 kd 30-35 kd Monomer trimer • Small G proteins have many key mechanistic and structural motifs in common with the Gα subunit of the heterotrimeric G proteins 42 proteins are low-molecularThe GTPase cycle •RAS-family weight guanine-nucleotide-binding proteins. – inactive when bound to GDP – active when bound to GTP •Regulation of this molecular: through a GDP-GTP cycle – guanine nucleotide-exchange factors (GEFs) • catalyse the exchange of GDP for GTP Inactive form – GTPase-activating proteins (GAPs) • increase the rate of GTP hydrolysis to GDP Active form •In the case of RHO proteins, another layer of regulation is provided by RHO–GDPdissociation inhibitors (RHOGDIs), which sequester RHO away from the GDP–GTP cycle. •GTPases interact with various effector proteins, which influence the activity and/or localization of these effectors; this ultimately influences cell-cycle progression 43 Nature Reviews Molecular Cell Biology 5, 355-366 (May 2004) RAS signaling 44 Nature Reviews Molecular Cell Biology 13, 39-51 (January 2012) 14.4 Many elements recur with variation in different signal-transduction pathways • Signal transduction has many common themes –Protein kinases are central to many signal-transduction pathways •protein kinases often phosphorylate multiple substrates and able to generate a diversity of responses –Second messengers participate in many signaltransduction pathways –Specialized domains that mediate specific interactions are present in many signaling proteins •pleckstrin homology domain: facilitate protein interactions with the lipid PIP3 •SH2 domain: mediate interactions with polypeptides containing phosphorylated tyrosine residues •SH3 domain: interact with peptide sequences that contain multiple proline residues 45 14.5 Defects in signal-transduction pathways can lead to cancer and other diseases •Signal transduction pathways can malfunction, leading to disease such as cancer •For example: –Rous sarcoma virus is a retrovirus that cause sarcoma in chicken •v-src genes is necessary for viral replication – an oncogene and v-Src is a protein tyrosine kinase –Normal chicken muscle cell: c-src •Not induce cell transform proto-oncogene •Encoded a signal transduction protein: regulates cell growth 46 •c-Src –Tyrosine residue near the C-terminal •When phosphorylated, it bound intramolecularly by the SH2 –the linker between the SH2 domain and the protein kinase domain is bound by the SH3 domain –These interactions hold the kinase domain in an inactive conformation •v-Src –C terminal 19 amino acids are replaced (lacks tyrosine) –Always active and promote unregulated cell growth 47 When Things Go Wrong… •Not all oncogenes caused by viruses •A mutated form of Ras can cause cancer – Usually mutations lead to loss of hydrolysis activity –If GTP cannot be hydrolyzed to GDP, stuck in the “on” position, stimulating cell growth •Mutations of “tumor suppressor genes” can cause cancer – E.g., mutations in genes that code for phosphatases involved in EGF signal termination EGF signaling persists initiated, stimulating inappropriate cell growth 48 Monoclonal antibodies can be used to inhibit signal-transduction pathways activated in tumors •Mutated/overexpessed receptor tyrosine kinases often observed in tumors –Epidermal-growth-factor receptor (EGFR) is overexpressed in some human epithelial cancers, including breast, ovarian, and colorectal cancer –Some EGFR can dimerize and send growth signal even in absence of EGF • Cetuximab (Erbitux) an antibody used therapeutically to prevent dimerization in colorectal cancer –EGFR family member, Her2 overexpressed in~30% of breast cancers •Trastuzumab (Herceptin) an antibody used to inhibit breast cancer 49 Protein kinase inhibitors may be effective anticancer drugs •Protein kinase inhibitors as anticancer drugs – Chronic myelogenous leukemia (CML,慢 性骨髓性白血病) often due to chromosomal defect where parts of chromosomes 9 and 22 are translocated, causing overexpression of a kinase (BcrAbl kinase) • An inhibitor specific for this kinase, Gleevec (STI-571, imatinib mesylate) very effective treatment Fig 14.33 The formation of the bcr-abl gene by translocation 50 DNA Damage and Repair 慢性骨髓性白血病 (chronic myelogenous leukemia) 的病生理機轉最 重要的就是第9對以及第22對染色體轉位, t(9:22),又稱為費城染色體(Philadelphia Chromosome)。 這種染色體轉位會造成原來位在第9對染色 體的Abelson (ABL) proto-oncogene接到 第22對染色體的breakpoint cluster region (BCR) 基因上,形成BCR-ABL chimeric 基因。正常的ABL 基因在轉錄轉 譯後產生的tyrosine kinase會受到嚴密的 調控;但發生費城染色體所形成的BCR-ABL fusion基因則失去正常的調控機轉,造成 tyrosine kinase過度表現的情形。在慢性 骨髓性白血病患者,有大於 90% 的患者會 有費城染色體,有大於 95% 的患者可以利 用PCR的方式找到BCR-ABL 基因。 51 Cholera霍亂and whooping cough百日咳are due to altered G-protein activity •Chloera toxin- Chloeragen is secreted by the intestinal bacterium Vibrio cholerae –Two functional unit– α β subunit that binds to GM1 ganglisoides of the intestinal epithelium –Catalytic A subunit that enters the cells •A subunits catalyzes the covalent modification of a Gαs protein – The α subunit is modified by the attachment of an ADP-ribose to an arginine – Stabilizes the GTP-bound form of Gαs active form continuously activates protein kinase A opens a chloride channel and inhibit sodium absorption •whooping cough – pertussis toxin from Bordetella pertussis –Adds an ADP-ribose moiety to Gαi, inhibit adenylate cyclase, close Ca2+ channel, open K+ channel, function “off” 52