受體與細(xì)胞功能調(diào)控第四次

cAMPpathwayPhosphatidylinositolpathwayGs、Gi、G12Gq

Adenylate

cyclase(AC)PhospholipaseC(PLC)ProteinkinaseA(PKA)Hydrolysisof4,5-diphospho-IntracellularcAMP

phosphatidyl

inositol(PIP2)Proteinphosphorylation

IP3（Ca2+）+DAGBiologicaleffectBiologicaleffect

twopathwaysSignaltransductionviaG-proteinSection7.SignaltransductionviaG-protein

ThePhosphoinositolpathway5.二脂肪酰甘油脂(diacyl-glycerol,DAG)和蛋白激酶C(PKC)PI-4,5-P2IP3+DAG

磷酸化后參予

PIP2的再生成循環(huán)

水解產(chǎn)生

花生四烯酸

(arachidonicA)

PKC(i)DAG?PKC(a)及前列腺素

(prostaglandin)分布：各種組織，其中腦、血癌細(xì)胞量最高

PKC的結(jié)構(gòu)和亞型基本結(jié)構(gòu)：經(jīng)典或普通PKC有四個保守區(qū)段

C1C2C3C4

DAG結(jié)合區(qū)

ATP結(jié)合位點鈣結(jié)合位點

催化活性部位亞型分類：

A類(經(jīng)典或普通PKC)鈣依賴性，DAG依賴性

B類(新PKC)無C2，無鈣依賴性

C類(不典型PKC)無C2，缺一半C1，無鈣及DAG依賴性每一類又分若干亞型以希臘字母區(qū)分PKC的活化及作用活化：假底物（pseudosubstrate)與PKC結(jié)合時，PKC處于非活性狀態(tài)。DAG或Phorbol與PKC的調(diào)節(jié)功能域結(jié)合時，PKC發(fā)生構(gòu)象(conformation)改變，假底物脫落，PKC激活

主要作用：催化多種蛋白磷酸化包括某些膜受體、激酶、收縮蛋白、代謝酶、轉(zhuǎn)錄因子、基因表達(dá)及蛋白合成有關(guān)的酶等ActivationofPKCPseudosubstratedomain:asequenceof18-19aminoacidlocatedinDAGbindingdomain.Atsilence,pseudosubstratebindwithcatalyticdomain.DAGbindingdissociatesthepseudosubstrate,PKCisactivated.ArtificialpseudosubstratecaninhibittherespectivePKC.PKC構(gòu)效關(guān)系PKC構(gòu)效關(guān)系和生理意義有待深入研究體內(nèi)PKC的功能調(diào)節(jié)對正常細(xì)胞的調(diào)節(jié)作用：活化生長因子對細(xì)胞粘附和分化的影響：對細(xì)胞間的信息交流的影響：生長、分化、抑制調(diào)節(jié)生長密度對原癌基因的表達(dá)的調(diào)節(jié)：限速步驟對基因表達(dá)的調(diào)節(jié)：對細(xì)胞核功能的調(diào)節(jié)：核蛋白激酶

PKC的反饋調(diào)節(jié)

PKC活化對細(xì)胞生長起正調(diào)節(jié)作用負(fù)反饋的調(diào)節(jié)：磷脂肌醇的降解、受體含量的負(fù)調(diào)節(jié)受體與配基的親和力降低、抑制受體酪氨酸磷酸化PKC和PKA的“對話”(crosstalking)Ca2+-CaMcAMPPKA活化

封閉PIP2水解

DAGPKCPKC與PKA途徑對細(xì)胞增殖和生長有相反作用

ConclusionofSignalTransductionofG-proteincoupledreceptorCascadeFourclassesofmembranousreceptorsChapter6

Section1Theother3kindsofmembranousreceptors1.Receptorkinases(Kinase-containing1TMSreceptors)

Basic

structure：

Extra-membraneregion:

Ligandbindingdomain

Transmembranousregion

Intramembranousregion:AkinasedomaininvolvingATPbindingsite.Basicfunction:Ligand+Receptor

Conformationalchangeofreceptor

(conformation構(gòu)象andconfiguration構(gòu)型)

ATPbindingsiteexposed，ATPboundtoreceptor

Kinasedomainphosphorylatedandactivated

Substrateincytosol

phosphorylated(Tyr,Ser/Thrresidues)orcGMPformation

CellresponseSubclasses(accordingtokinaseproperty)

Tyrosinekinase(

phosphorylationof

substrate

containingTyr)Serinekinase(

phosphorylationof

substrate

containingSer/Thr)Guanosine

cyclase(GTP

cGMP)蛋白質(zhì)酪氨酸激酶(PTK)

激酶結(jié)構(gòu)域的特異性:分為亞組,有10種以上。識別專一底物中的酪氨酸殘基。功能域有強大的生理催化活性轉(zhuǎn)導(dǎo)細(xì)胞外的生長和分化；細(xì)胞對胞內(nèi)氧化還原勢的響應(yīng)等等功能。1)ReceptorTyrosinekinase(RTK

)Intrinsicagonist:vairousgrowthfactors(notgrowthhormone)agonistreceptorRTK

phosphorylationofsubstrateTyr

Classification:accordingtoextramembranestructure&

function

(1)Epidermalgrowthfactor(表皮生長因子)receptor(EGFR)

(2)Insulinreceptor(IR)(involvedingrowthpromotion)

(3)Plateletderivedgrowthfactor(血小板衍生生長因子)receptor(PDGFR)(involvedinchemotaxisandproliferationofconnectivetissue)

(4)Nervegrowthfactor(神經(jīng)生長因子)receptor(NGFR)

Nosubtype,curvatureofScatchardplotisduetonegativecooperativityEpidermalgrowthfactorreceptors(EGFR)：

extra-membranousportionhas2cycteine-enrichedstructure

CCHER2,HER3,HER4:homologsofEGFRHER2:homologsofEGFR在正常細(xì)胞中，只含有少量的HER-2。HER-2在調(diào)控正常細(xì)胞的生長和發(fā)育和分化。

HER2過度表達(dá)組織：乳腺、卵巢、肺、胰腺及胃腸道腫瘤細(xì)胞。HER2與HER3，HER4之間的異二聚體被活化，促進細(xì)胞的增殖。診斷：早期；HER2過度表達(dá)，病情發(fā)展快；預(yù)后化療差。治療：曲妥珠單抗（赫賽汀）與RTK的細(xì)胞外域結(jié)合,可誘導(dǎo)受體內(nèi)化,引起HER-2下調(diào),使細(xì)胞周期阻滯；與受體作用阻滯HER-2的過度活化。InsulinReceptors(IR):

Composedof、chainsconnectedbydisulfidebond

Othermembers:

IGF-IR(Insulin-likeGFR)；

HGFR(HepatocyteGFR)tworeceptorsareconnectedbyadditionaldisulfidebondtoformastabledimer

Plateletderivedgrowthfactorreceptor(PDGFR)

(proliferationofconnectivetissure):

Extracellularportioncomposedof2~7immunoglobulin-likestructureLigand

Othermembers:

FGF(Fibroblastgrowthfactor)R

VEGF(Vascularendotheliumgrowthfactor)R

CSF-1(Colonystimulatinggrowthfactor1)R

KGF(Keratinocytegrowthfactor)R

SCF(Stemcellgrowthfactor)R

Nervegrowthfactorreceptor(NGFR,TrkA)：

ALeu,Cys-richstructurelocatedatextramembranousportion

Othermembers:Brainderivedneurotrophicfactor(BDNF)receptor,TrkB

Neurotrophins(NTs)receptor,TrkC(NT3)

Glialcelllinederivedgrowthfactor(GDNF)receptor(GFR1,Ret)Ligandbinding

Glialcelllinederivedgrowthfactor(GDNF)receptorisinfactnotaRTK

LNGFR

(NGFR-P75):BindtoNGF、BDNF、NTswithlowaffinityNotRTKbutbelongtothekinase-linkedsingleTMSclassGDNFbindingtriggerstheassociationofGFR1withRet.RetisatransmembraneproteinTKofabout150kDa.ThereforeGDNFRisakindofTKlinkedreceptorbutnotaTKcontainingreceptor.蛋白質(zhì)相互作用的調(diào)控元件

（molecularbindingdomain）蛋白質(zhì)分子中的特殊結(jié)構(gòu)域，信號分子相互識別的部位。類別-SH2結(jié)構(gòu)域(srchomology2domain)介導(dǎo)信號分子與磷酸化的酪氨酸蛋白分子結(jié)合-SH3結(jié)構(gòu)域(srchomology3domain)介導(dǎo)信號分子與富脯氨酸蛋白分子結(jié)合-PH結(jié)構(gòu)域(pleckstrinhomologydomain)-PTB結(jié)構(gòu)域(proteintyrosinebindingdomain)識別磷酸酪氨酸基序SH2結(jié)構(gòu)域(SrcHomology2domain):識別作用

酪氨酸蛋白和含SH2結(jié)構(gòu)域蛋白的相互作用:

酪氨酸的磷酸化是高親和力的前提，+1、+2、+3位置上的氨基酸和結(jié)合特異性有關(guān)。

膜內(nèi)酪氨酸激酶活性結(jié)構(gòu)域膜內(nèi)其它磷酸化部位：酪氨酸激酶的活性提高

酪氨酸激酶的活性降低

磷酸化和受體內(nèi)移有關(guān)

以EGFR為例:

PKC磷酸化，磷酸化的結(jié)果使受體的酪氨酸

作用減弱；

MAP激酶磷酸化，和受體的內(nèi)移有關(guān)其它磷酸化部位連接蛋白

GRb2(growthfactorreceptor-boundprotein)：GRb2/Sos復(fù)合物，激活的受體與其下游的信號轉(zhuǎn)導(dǎo)分子相連接。它有一個SH2域，其側(cè)面是兩個SH3域。它與EGF-R,胰島素受體(IR),胰島素受體底物-1(IRS-1)信號分子等等的磷酸酪氨酸殘基結(jié)合。Ligand+receptor

Receptordimerization

Conformationalchange

Kinaseauto-phosphorylated(perhapsbycrossing)

Receptor

kinaseactivation

Substrateincytosol

phosphorylated

attyrosineresidueandactivated

MAPKpathwaycascade

CellfunctionchangeMainpathwayofsignaltransductionandfunctionofRTK1.Promotecellgrowth,developmentanddifferentiationofrelatedtissueEGF+EGFR1min,receptorkinase

phosphorylated1hr,cytosolproteinphosphorylated2hr,DNAsynthesisinnucleus

Ras

-GDPRas-GTPRTKThecentralmoleculeofMAPKpathwayisRas.RasislowMWG-proteinandisaproto-oncogen.Thereare2formsofRas,Ras-GDPandRas-GTP.Ras-GDPsuppressesandRas-GTPpromotessignaltransduction.RTKdoesnotactonRasdirectly.Ras

-GDPRas-GTPRTKWhenRTKisphosphorylated,itcanberecognizedbyproteinscontainingSH2-domain.ForEGFR,itisGrb2,forIR,itisIRS-1.Grb2andIRS-2(throughGrb2)hasnoenzymeactivity,butactsasmediator,knownas“adaptorproteins”.Grb2activatesRas-GDS(GDPdissociationstimulator),whichturnsRas-GDPtoRas-GTPTherefore,duringRTKphosphorylation,Ras-GDPturnstoRas-GTPIRS-1Grb2Ras

-GDS

GDPGTPRas

-GDPRas-GTPRTKRas-GTPactivatedRaf,whichisaSer/Thr

kinase.ActivatedRafactivatesMAPKK(MAPkinase

kinase).MAPKKactivatesMAPK,whichisknownasmitogen-activatedproteinkinase,alsoknownasERK(extracellularsignalregulatedkinase).Aseriesofmitosis-relatedbiochemicaleventsinnucleusisactivated.(activationofAp-1andSap-1whichinturnactivatestranscriptionalfactorsandotherkinases)IRS-1Grb2Ras

-GDS

GTPGDP

Raf

MAPKK

ERK

NucleuseventsRas

-GDPRas-GTPRTKGAP(GTPase-activatingprotein)canstimulatetheGTPaseactivityofRas.DuringsilenceofRTK,GAPactivatesGTPaseactivityofRasandturnsRas-GTPtoRas-GDP.PhosphorylationofRTKsuppressestheactivityofGAPwhichinturnsuppressesthehydrolysisofRas-GTP.IRS-1Grb2Ras

-GDS

GTPGDP

Raf

MAPKK

ERK

GAPPi(Phosphorylated)(-)NucleusGeneexpressionregulationras-GTPase的主要功能是控制

MAPK級聯(lián)反應(yīng)

ras-GTPase。它是一個對酪氨酸激酶與絲氨酸/蘇氨酸激酶之間的信號轉(zhuǎn)導(dǎo)通路的分子轉(zhuǎn)換器，這些通路導(dǎo)致細(xì)胞分化或者增殖。許多人類的腫瘤有被激活的ras癌基因，說明了ras是細(xì)胞分化或者增殖的強力調(diào)節(jié)者。

酪氨酸激酶抑制劑（TKI）具有抑制酪氨酸激酶活性的藥物的總稱。靶向PDGFR的酪氨酸激酶抑制劑有舒尼替尼和索拉菲尼，臨床最常用的腫瘤藥物之一。作用機制：通過抑制RAF/MEK/ERK信號傳導(dǎo)通路，直接抑制腫瘤生長。通過抑制PDGFR和VEGFR而阻斷腫瘤新生血管的形成，間接抑制腫瘤細(xì)胞的生長。SignaltransductionviareceptorkinasesthreepathwaysAnotherimportantpathwayofsignaltransductionfromRTK:PI3K-AKT-mTOR

PI3-K（磷脂酰肌醇-3激酶）的細(xì)胞功能可能是它參與有絲分裂信號的轉(zhuǎn)導(dǎo)。PI3-K/PKB/mTOR

PI-3-K：特異催化細(xì)胞膜上PIP2的肌醇環(huán)3‘OH磷酸化，生成PIP3（secondmessage）

AKT(PKB):癌基因v-akt編碼的Akt蛋白與PKB同源

mTOR：哺乳動物雷帕霉素靶蛋白(mammaliantargetofrapamycin)腫瘤：PI3K,Akt

過度活躍

抑制蛋白：PTEN：抑制Akt

TSC1/TSC2：抑制Akt、mTOR

重要性：腫瘤進展和治療靶點Ｇ-蛋白的后續(xù)信號轉(zhuǎn)導(dǎo)（三）其它信號通路

趨化因子受體：信號傳導(dǎo)通路&MAPK/ERK1/2途徑細(xì)胞繁殖、突觸傳遞&

PI-3K/AKT途徑細(xì)胞存活率、粘附、趨化。Otherpossiblepathwaysofmitosis-activationbyphosphorylationofRTK

ThroughCdc2(aCell-division-cyclekinase)H1磷酸化(染色質(zhì)縮合)、DNA結(jié)合蛋白磷酸化(啟動延長)ThroughS6KI(aribose-ribosomekinase)

有絲分裂的重要激酶OtherphosphorylationsitesofRTKSomesitesmaybephosphorylatedbyPKC:decreaseactivityofRTKSomesitesmaybephosphorylatedbyMAPK:relatedtointernalizationSomesitesmaybephosphorylatedbyCaMKII:decreaseactivityofRTKSomesitesmaybindPLC()whenRTKisphosphorylatedleadingtophosphorylationandactivationofPKCthroughDAGThesesitesthusformanetworkbetweenRTKandotherreceptors,theirpracticalsignificanceneedsfurtherexploration絲氨酸/蘇氨酸磷酸化激酶2)Receptorproteinserine/

threonine

kinase(PSTK)

Distribution:

widelydistributedinmanytissues

Structure:Eachreceptorcomposedof2-3subunitsOnlytheheterpolymerhashighaffinitytoagonist.BasicstructuresimilartoRTK,butPSTKreplaceRTK

Function:promotecelldifferentiation

Inhibitexcessivecellproliferation

Members:Transforminggrowthfactorreceptor(TGFR,TR)

BMP(bone

morphogeneticprotein)Receptors(osteogenesisandneurogenesis)

ActivinReceptors(FSHproduction)

GDFReceptorss(chondrogenesisindevelopinglimbs)

TGF+TR

ReceptorPSTK(proteinSer/Thr

kinase)phosphorylation

Smadcontactwithreceptorandphosphorylated

G2Signaltransduction:ThroughSer/Thr

kinase(PSTK)Oligomerformation,kinase

phophorylatedandactivatedSmadisaprotein.MAD:productofDrosophila(果蠅)gene(mothersagainstDrosophila)SMAD:MADhomologuesbecausetheirmutationcausessmallbodysizeClassification:accordingtostructure&functionR-Smads(Receptor-regulatedSmads):Smad1、2、3、5、8subtype，C端含有磷酸化位點Co-Smads(Common-partnerSmad):Smad4，無磷酸化位點InhibitorySmads:Smad6、7，無磷酸化位點，有受體結(jié)合位點（與受體結(jié)合，阻止R-Smads

與受體結(jié)合）R-Smads與Smad4形成活性復(fù)合物，調(diào)控基因表達(dá)的生物學(xué)作用Phosphorylated

Smadmovetonucleus

bindtonuclearDNA(atpromotorregion)

affectgeneexpressionSequenceofDNA:GTCTAGAC

(atleast2copiesofGTCT)

PromotionofcelldifferentiationStimulatingsubtypesofphosphorylated

Smad:

Smad1,2,3,5,8bindtothesequenceatthepromotorregionResultofbinding:stimulategeneexpression

Inhibitionofhyper-proliferation

Inhibitorysubtypesofphosphorylated

Smad:

Smad6and7Possiblemechanism:

Smad6and7havenostimulatingeffect,butcancompetethethetargetmoleculeswiththestimulatingSmads

Smad6and7caninteractwithsomeotherproteinandactsontranscriptionalfactorstoinhibitcellcycleatG1stage3.MembranousG-cyclase

AngiotensinPKGcGMP

IP3NOCa2+Ca2+-CaMCaMKII