浙师大细胞生物学2ppt课件

Chapter 11:Cell-to-Cell SignalingA:Overview of Extracellular Signaling B:Signaling via Hydrophobic MoleculesC:Signaling via Ion ChannelsD:Signaling via G-Protein-Coupled ReceptorsE:Signaling via Receptor Tyrosine KinasesF:MAP Kinase Pathways G:Interaction and Regulation of Signaling Pathways A:Overview of Extracellular Signaling Communication by extracellular signals usually involves six steps:(1)synthesis and(2)release of the signaling molecule by the signaling cell;(3)transport of the signal to the target cell;(4)detection of the signal by a specific receptor protein;(5)a change in cellular metabolism,function,or development triggered by the receptor-signal complex;and(6)removal of the signal,which often terminates the cellular response.1.General schemes of intercellular signaling in animals.In animals,signaling by extracellular,secreted molecules can be classified into three types based on the distance over which the signal acts.In addition,certain membrane-bound proteins on one cell can directly signal an adjacent cell.In endocrine signaling,signaling molecules,called hormones,act on target cells distant from their site of synthesis by cells of endocrine organs.In paracrine signaling,the signaling molecules released by a cell only affect target cells in close proximity to it.In autocrine signaling,cells respond to substances that they themselves release.Many growth factors act in this fashion,and cultured cells often secrete growth factors that stimulate their own growth and proliferation.(a-c)Cell-to-cell signaling by extracellular chemicals occurs over distances from a few micrometers in autocrine and paracrine signaling to several meters in endocrine signaling.(d)Proteins attached to the plasma membrane of one cell can interact directly with receptors on an adjacent cell.2.Receptor Proteins Exhibit Ligand-Binding and Effector Specificity The signaling molecule(hormone,pheromone,or neurotransmitter)acts as a ligand,which binds to,or fits,a site on the receptor(intracellular receptors;and cell-surface receptors).Binding of a ligand to its receptor causes a conformational change in the receptor that initiates a sequence of reactions leading to a specific cellular response.3.Hormones Can Be Classified Based on Their Solubility and Receptor Location Most hormones fall into three broad categories:(1)small lipophilic molecules that diffuse across the plasma membrane and interact with intracellular receptors;and(2)hydrophilic or(3)lipophilic molecules that bind to cell-surface receptors.Recently,nitric oxide,a gas,has been shown to be a key regulator controlling many cellular responses.Some hormones bind to intracellular receptors;others,to cell-surface receptors.(a)Steroid hormones,thyroxine,and retinoids,being lipophilic,are transported by carrier proteins in the blood.After dissociation from these carriers,such hormones diffuse across the cell membrane and bind to specific receptors in the cytosol or nucleus.The receptor-hormone complex then acts on nuclear DNA to alter transcription of specific genes.(b)Polypeptide hormones and catecholamines(e.g.,epinephrine),which are water soluble,and prostaglandins,which are lipophilic,all bind to cell-surface receptors.This binding triggers an increase or decrease in the cytosolic concentration of second messengers(e.g.,cAMP,Ca2+),activation of a protein kinase,or a change in the membrane potential.Hydrophobic molecules Ion channels G-protein-coupled receptors Enzymes(e.g.RTKs).In many cases,the signal continues to propagate within the cell and often reaches nuclear DNA to express proteins.4.The external signal may enter a cell via four major pathways:Major pathways for signals to enter a cell.Cell-Surface Receptors Belong to Four Major Classes Section B:Signaling via Hydrophobic MoleculesHydrophobic molecules can move in and out of cells by passing through lipid bilayers.Nitric oxide,arachidonic acid and steroids have been shown to play important roles in cell signaling.Signaling with NO or arachidonic acid.Unlike most signaling cascades which occur within the same cell,newly generated NO or arachidonic acid diffuses to act on target molecules in neighboring cells.Nitric oxide(NO)is produced from the following reaction:where NOS represents NO synthase.After NO is generated in a cell,it diffuses to act on target molecules in neighboring cells.NO may stimulate soluble guanylyl cyclase to produce cGMP which regulates several enzymes and ion channels.In smooth muscle,an important action of cGMP is to induce muscle relaxation.Normally,cGMP will soon be converted into GMP by phosphodiesterase.The well known drug for impotence,Viagra(sildenafil citrate),inhibits phosphodiesterase.Nitric oxideArachidonic acid is generated from phospholipid hydrolysis catalyzed by phospholipase.After diffusing to target cells,arachidonic acid may activate protein kinase,resulting in phosphorylation of target molecules.Many of its target molecules are involved in learning and other neuronal activities.Arachidonic acidGeneration of arachidonic acid from phospholipid by phospholipase A2(PLA2).SteroidsThe major role of steroids is to regulate transcription,since many steroid receptors are transcription factors.A transcriptional activation mechanism by steroid receptor(SR).Steroid-bound SR may recruit SRC(steroid receptor coactivator)and histone acetyltransferases to stimulate transcription of the target gene.Section C:Signaling via Ion ChannelsIon channels are membrane proteins that allow ions to pass through.In terms of ion selectivity,they are classified as calcium channels,sodium channels,potassium channels,etc.In terms of gating,they may be classified as voltage-gated channels,ligand-gated channels,etc.is to regulate membrane potential.However,the calcium ion also plays important roles in other cellular functions,since many enzymes are calcium-dependent.Through voltage-activated Ca2+channels whose opening probability depends on the membrane potential.Through IP3-sensitive Ca2+channels whose opening probability is regulated by IP3(inositol trisphosphate).The opening of Ca2+stores in the endoplasmic reticulum may result in calcium waves.The major role of sodium and potassium ionsThe major role of G-protein-coupled receptors is to transmit signals into the cell.They are characterized by seven transmembrane segments.This class of membrane proteins can respond to a wide range of agonists,including photon,amines,hormones,neurotransmitters and proteins.Some agonists bind to the extracellular loops of the receptor,others may penetrate into the transmembrane region.Section D:Signaling via G-protein-Coupled Receptors1.G-Protein-Coupled Receptors 2.G Proteins 3.Effectorsgpcr.org G-Protein-Coupled Receptor Database1.G-protein-Coupled ReceptorsThe major role of G-protein-coupled receptors is to transmit signals into the cell.The full name of G protein is GTP-binding protein because in the active state it binds to GTP(guanosine triphosphate).2.G ProteinsThere are two types of G proteins:heterotrimeric G proteins and monomeric G proteins(or small G proteins).Ras and Ran are small G proteins.G-protein-coupled receptors are coupled to heterotrimeric G proteins.The structure of heterotrimeric G protein,consisting of three subunits:a,b and g.In the inactive state(GDP)Based on the differences in their genes,20 a,6 b and 12 g subunits have been identified.Their molecular weights are in the following ranges:a subunit:39-46 kD b subunit:35-39 kD g subunit:8 kD Cycling of G protein between active and inactive statesInteraction between Ga and the agonist-stimulated receptor causes the release of GDP.GTP then binds to the empty site because its concentration in the cell is higher than GDP.3.G Protein EffectorsEffectors are the target molecules of G protein a or bg subunit.The table lists the major effectors of the a subunit.(+)means activate and(-)means inhibit.Upon binding of the cholera toxin,the Ga-bound GTP cannot be converted into GDP so that the G protein remains in the active state.By contrast,pertussis toxin prevents GDP release from the a subunit so that the G protein is locked in the inactive state.ClassGene VariantEffectorToxin SensitivityGsa as(s)a as(L)(+)Adenylyl cyclase(+)Ca2+channel(-)Na+channelCholeraa aolf(+)Adenylyl cyclaseCholeraGia ai1a ai2a ai3(-)Adenylyl cyclase(-)Ca2+channel(+)K+channelPertussisa aoa a aob(-)Ca2+channel(+)Phospholipase C(+)Phospholipase A2Pertussisa at1 a at2(+)cGMP phosphodiesteraseCholera and Pertussisa ag(+)Phospholipase CPertussisa a2(-)Adenylyl cyclaseGqa aq a a11 a a14a a15a a16(+)Phospholipase CG12a a12 a a13?(a)Before agonist binding to the G-protein-coupled receptor,the three subunits of G protein are bound together.(b)The agonist binding causes interaction between the G-protein-coupled receptor and the G protein.(c)Their interaction results in the dissociation between a and bg subunits of the G protein.The separated a and/or bg subunits may then interact with effectors.Signaling via G-protein-Coupled ReceptorsThe bg subunits may act on adenylate cyclase,phospholipase A2,phospholipase C,ion channels,calcium ATPase etc.Adenylate cyclase Adenylate cylase catalyzes the conversion of ATP to cAMP,which is an important second messenger.Second messengers are the signaling molecules generated by the stimulation of cell-surface receptors.For example,cAMP,arachidonic acid,DAG and IP3 generated by the activation of G-protein-coupled receptors are second messengers.The agonists which activate G-protein-coupled receptors are first messengers.Signaling with cAMPA cAMP/PKA signaling pathway.PKA consists of two catalytic subunits and two regulatory subunits.cAMP binds to two sites on each regulatory subunit.Binding of four cAMP molecules causes the release of free and active catalytic subunits,which may phosphorylate serine and threonine residues on target proteins.In this figure,the active subunit phosphorylates a CREB protein,resulting in transcription.Phospholipase C(PLC)also cleaves phospholipids,generating diacylglycerol(DAG)and IP3(inositol 1,4,5-trisphosphate).IP3 can activate an IP3-sensitive Ca2+channel in endoplasmic reticulum.Signaling with DAG and IP3RTKs are the second major type of cell-surface receptors.The ligands for RTKs are soluble or membrane-bound peptide/protein hormones including nerve growth factor(NGF),platelet-derived growth factor(PDGF),fibroblast growth factor(FGF),epidermal growth factor(EGF),and insulin.Binding of a ligand to this type of receptor stimulates the receptors intrinsic protein-tyrosine kinase activity,which subsequently stimulates a signal-transduction cascade leading to changes in cellular physiology and/or patterns of gene expression.RTK signaling pathways have a wide spectrum of functions including regulation of cell proliferation and differentiation,promotion of cell survival,and modulation of cellular metabolism.Section E:Signaling via Receptor Tyrosine Kinases(RTKs)Activation of RTKs transmit a hormone signal to Ras.1.Ligand Binding Leads to Autophosphorylation of RTKs 2.Ras Belongs to the GTPase Superfamily of Intracellular Switch Proteins Ras is a GTP-binding switch protein that,like the G a subunits in different G proteins,alternates between an active on state with a bound GTP and an inactive off state with a bound GDP.Ras activation is accelerated by a protein called guanine nucleotide exchange factor(GEF),which binds to the Ras GDP complex.3.An Adapter Protein and GEF Link Most Activated RTKs to Ras Activation of Ras following binding of a hormone(e.g.,EGF)to an RTK.The adapter protein GRB2 binds to a specific phosphotyrosine on the activated RTK and to Sos,which in turn interacts with the inactive Ras GDP.The guanine nucleotide exchange factor(GEF)activity of Sos then promotes formation of the active Ras GTP.The phosphate group of phosphotyrosine(phosphorylated tyrosine)can form several hydrogen bonds with the SH2(Src homology 2)domain of some proteins involved in the signaling via tyrosine kinases.Receptor Tyrosine KinasesDomain structure of some SH2-containing proteins.The epidermal growth factor(EGF)peptide induces cellular proliferation through the EGF receptor,which has a tyrosine kinase cytoplasmic domain,a single transmembrane domain and an extracellular domain involved in EGF binding and receptor dimerization.Inhibitors of the EGF receptor are being pursued as potential cancer therapies and EGF may stimulate wound healing.Mutation of the EGF receptor has been associated with cancer in humans.The proliferative effects of EGF are signaled through several pathways.Binding of EGF results in EGF receptor dimerization,autophosphorylation of the receptor,and tyrosine phosphorylation of other proteins.The EGF receptor activates ras and the MAP kinase pathway,ultimately causing phosphorylation of transcription factors such as c-Fos to create AP-1 and ELK-1 that contribute to proliferation.Activation of STAT-1 and STAT-3 transcription factors by JAK kinases in response to EGF contributes to proliferative signaling.Phosphatidylinositol signaling and calcium release induced by EGF activate protein kinase C,another component of EGF signaling.Crosstalk of EGF signaling with other pathways make the EGF receptor a junction point between signaling systems.Platelet Derived Growth Factor(PDGF)plays a critical role in cellular proliferation and development.The biologically active form is a dimer formed from the A and B chains.PDGF is active to a differing degree depending on which dimer is formed(AA,AB,or BB).The PDGF Receptor(PDGFR)is also a dimer and can form from the combination of the alpha and beta chains in any order(alpha-alpha,alpha-beta,beta-beta).The PDGFR dimer is only formed after ligand binding so the alpha/beta composition of the receptor can be influenced by the form of PDGF that is present.Upon binding of ligand the PDGFR is tyrosine phosphorylated and leads to the phosphorylation of several other cellular proteins.Receptor tyrosine kinases(RTKs),which bind to peptide/protein hormones,may exist as dimers or dimerize during binding to ligands.Ligand binding leads to activation of the kinase activity of the receptor and autophosphorylation of tyrosine residues in its cytosolic domain.The activated receptor also can phosphorylate other protein substrates.Ras is an intracellular GTPase switch protein that acts downstream from most RTKs.Like Gsa,Ras cycles between an inactive GDP-bound form and active GTP-bound form.Ras cycling requires the assistance of two proteins,GEF and GAP,whereas Gsa cycling does not.SUMMARY of RTKsUnlike GPCRs,which interact directly with an associated G protein,RTKs are linked indirectly to Ras via two proteins,GRB2 and Sos.The SH2 domain in GRB2,an adapter protein,binds to specific phosphotyrosines in activated RTKs.The two SH3 domains in GRB2 then bind Sos,a guaninenucleotide exchange factor,thereby bringing Sos close to membrane-bound Ras GDP and activating its exchange function.Binding of Sos to inactive Ras causes a large conformational change that permits release of GDP and binding of GTP.Normally,Ras activation and the subsequent cellular response is induced by ligand binding to an RTK.However,in cells that contain a constitutively active Ras,the cellular response occurs in the absence of ligand binding.All Ras-linked RTKs in mammalian cells appear to utilize a highly conserved signal-transduction pathway in which the signal induced by ligand binding is carried via GRB2 and Sos to Ras,leading to its activation.Activated Ras then induces a kinase cascade that culminates in activation of MAP kinase.This serine/threonine kinase,which can translocate into the nucleus,phosphorylates many different proteins including transcription factors that regulate expression of important cell-cycle and differentiation-specific proteins.Activation of MAP kinase in two different cells can lead to similar or different cellular responses,as can activation in the same cell by stimulation of different RTKs.Section F:MAP(Mitogen-activated protein)Kinase Pathways1.Signals Pass from Activated Ras to a Cascade of Protein KinasesA remarkable convergence of biochemical and genetic studies has revealed a highly conserved cascade of protein kinases that operate in sequential fashion downstream from activated Ras:1.Activated Ras binds to the N-terminal domain of Raf,a serine/threonine kinase.2.Raf binds to and phosphorylates MEK,a dual-specificity protein kinase that phosphorylates both tyrosine and serine residues.3.MEK phosphorylates and activates MAP kinase,another serine/threonine kinase.4.MAP kinase phosphorylates many different proteins,including nuclear transcription factors,that mediate cellular responses.In unstimulated cells,most Ras is in the inactive form with bound GDP;binding of a growth factor to its RTK leads to formation of the active Ras GTP.A signaling complex then is assembled downstream of Ras,leading to activation of MAP kinase by phosphorylation of threonine and tyrosine residues separated by a single amino acid.Biochemical studies showed the signaling pathway appears to be a linear one:activated RTK Ras Raf MEK MAP kinase.Activation of Ras induces a kinase cascade 2.Ksr May Function as a Scaffold for the MAP Kinase Cascade Linked to Ras Two additional proteins,14-3-3 and Ksr,participate in the MAP kinase cascade downstream from Ras.Although the precise functions of these proteins are not yet known,they both appear to play important roles in forming protein complexes necessary for signaling from Raf to MAP kinase.In a resting cell prior to stimulation,Raf is present in the cytosol in an inactive conformation stabilized by a dimer of 14-3-3.Each 14-3-3 monomer binds to a phosphoserine residue in Raf,and the dimer appears to have a more general function in linking together signaling components through phosphoserine residues.Activation of Raf also requires Ksr,which contains binding sites for Raf,14-3-3,MEK,and MAP kinase.Ksr may function as an adapter protein,providing a scaffold for formation of a large signaling complex that continues to operate after Raf dissociates from Ras GDP.Although Ksr has a kinase domain,genetic studies in Drosophila have shown that this domain has a negative regulatory function,perhaps acting as part of a switch for regulating the turnover of components within the complex.Fuction of Ksr3.Phosphorylation of a Tyrosine and a Threonine Activates MAP Kinase Structures of MAP kinase in its inactive and active formBoth the phosphorylated tyrosine and threonine residues in MAP kinase interact with additional amino acids,thereby conferring an altered conformation to the lip region,which in turn permits binding of ATP to the catalytic site.The phosphotyrosine residue plays a key role in forming the binding site for specific substrate proteins on the surface of MAP kinase.Phosphorylation promotes not only the catalytic activity of MAP kinase but also its dimerization.The dimeric form of MAP kinase can be translocated to the nucleus where it regulates the activity of a number of nuclear localized transcription factors.4.Various Types of Receptors Transmit Signals to MAP Kinase Although yeasts and other single-celled eukaryotes lack RTKs,they have been found to possess MAP kinase pathways.And in different cell types of higher eukaryotes,stimulation of receptors other than RTKs also can initiate signaling pathways leading to activation of MAP kinase.The mating pathway in S.cerevisiae is a well-studied example of a MAP kinase cascade that is linked to G protein coupled receptors,in this case for two secreted peptide pheromones,the a and a factors.5.Multiple MAP Kinase Pathways Are Found in Eukaryotic CellsBoth yeasts and higher eukaryotic cells contain other functionally equivalent proteins,including the Jun N-terminal kinases(JNKs)and p38 kinases in mammalian cells and six yeast proteins.All these proteins are serine/threonine kinases that are activated in the cytosol in response to specific extracel