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1、Chapter 11:Cell-to-Cell Signaling,A: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:Over
2、view 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
3、 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
4、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
5、 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 gro
6、wth 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
7、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 cause
8、s 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
9、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
10、-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
11、 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 concentrat
12、ion 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 pro
13、teins.,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 Molecules,Hydrophobic molecules can move in and out of cells by passing through lipid bilayers.Ni
14、tric 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.,N
15、itric 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 mus
16、cle,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 oxide,Arachidonic acid is generated from phospholipid hydrolysis catalyzed
17、 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 acid,Generation of arachidonic acid from phospholipid by ph
18、ospholipase A2(PLA2).,SteroidsThe major role of steroids is to regulate transcription,sincemany steroid receptors are transcription factors.,Section C:Signaling via Ion Channels,Ion channels are membrane proteins that allow ions to pass through.In terms of ion selectivity,they are classified as calc
19、ium 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
20、.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 r
21、ole of sodium and potassium ions,The 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 pr
22、oteins.Some agonists bind to the extracellular loops of the receptor,others may penetrate into the transmembrane region.,Section D:Signaling via G-protein-Coupled Receptors,1.G-Protein-Coupled Receptors 2.G Proteins 3.Effectors,G-Protein-Coupled Receptor Database,1.G-protein-Coupled Receptors,The ma
23、jor 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 Proteins,There are two types of G proteins:heterotrimeric G proteins and monomeric G proteins(or sm
24、all 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
25、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 states,Interaction between Ga and the agonist-stimulated receptor causes the release of GDP.GTP then binds to the empty site becau
26、se its concentration in the cell is higher than GDP.,3.G Protein Effectors,Effectors 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 converte
27、d 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.,(a)Before agonist binding to the G-protein-coupled receptor,the three subunits of G protein are bound together.(b)Th
28、e 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 Receptors,The
29、 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
30、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 cAMP,A cAMP/PKA signaling pathway.PKA consists of two cat
31、alytic 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 phosphoryl
32、ates 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 IP3,RTKs are the second major type of cell
33、-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 re
34、ceptors 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 differen
35、tiation,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
36、 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
37、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 gua
38、nine 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.,Recept
39、or Tyrosine Kinases,Domain 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 r
40、eceptor 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
41、 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 proliferati
42、on.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 pathw
43、ays 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 dim
44、er 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 f
45、orm 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
46、 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 cycle
47、s 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 RTKs,Unlike GPCRs,which interact directly with an associated G protein,RTKs are linked indirectly to Ras via two proteins,GRB2 and S
48、os.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 cau
49、ses 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
50、.,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
