chapter 15 –signal transduction and g protein–coupled ... · pdf...

Chapter15– SignalTransductionandGProtein–CoupledReceptors

Signaltransduction?• Signaltransduction (alsoknownascellsignaling)isthe

transmissionofmolecular signals fromacell'sexteriortoitsinterior. Signals receivedbycellsmustbetransmittedeffectivelyintothecelltoensureanappropriateresponse.Thisstepisinitiatedbycell-surfacereceptors.

Chapter15– SignalTransductionandGProtein–CoupledReceptors

15.1SignalTransduction:FromExtracellularSignaltoCellularResponse15.2StudyingCell-SurfaceReceptorsandSignalTransductionProteins15.3GProtein–CoupledReceptors:StructureandMechanism15.4GProtein–CoupledReceptorsThatRegulateIonChannels15.5GProtein–CoupledReceptorsThatActivateorInhibitAdenylylCyclase15.6GProtein–CoupledReceptorsThatTriggerElevationsinCytosolicandMitochondrialCalcium

SignalTransductionandGProtein–CoupledReceptors

15.1SignalTransduction:FromExtracellularSignaltoCellularResponse• Allcellsrespondtoextracellularsignals/stimulithatactivateplasmamembraneorcytosolicreceptors.



• ActivatedreceptorsfunctionastranscriptionfactorsoractivateGproteinswitchesthatregulateavarietyofdownstreampathways orinducegenerationofintracellularsecondmessengers thatdoso.

How????



• ActivatedreceptorsfunctionastranscriptionfactorsoractivateGproteinswitchesthatregulateavarietyofdownstreampathways orinducegenerationofintracellularsecondmessengers thatdoso.

• Proteinphosphorylationbykinasesanddephosphorylationbyphosphatasesregulateproteinactivityinthecellularpathwaysandcanamplifyintracellularsignaling.

Overviewofcellsignaling

• Extracellularsignalingmolecules–synthesized,packagedintosecretoryvesicles,andsecretedbyspecializedsignalingcellswithinmulticellularorganisms

• Signal– producesaspecificresponseonlyintargetcellsexpressingreceptorproteinsthatbindthesignal

Hydrophobicsignals

vs.

Hydrophilicsignals

Howdifferent?

Typesofextracellularsignaling.

• Extracellularmoleculesignaling– threeclassifications;basedondistanceoverwhichthesignalacts:

• (a)Endocrine:(epinephrine,insulin)• Signalingmolecules– synthesizedandsecretedbysignalingcells(e.g.,cellsinendocrineglands)

• Transportedthroughthecirculatorysystem• Affectdistanttargetcellsexpressingthereceptor

• (b)Paracrine:(neurotransmitters,growthfactors)• Signalingmoleculessecretedbyacell– affectonlynearbytargetcellsexpressingthereceptor

• SomemaybindtoECM– releasedonlywhenECMisdegraded

• (c)Autocrine:(growthfactors)• Cellsrespondtosignalstheysecrete.(Tumorcellsmayoverproduceandrespondtogrowthfactors.)

• (d)Membraneproteinsignals: signalneighboringcellsbydirectcontactwithsurfacereceptors.

Howtoregulatethefunctionofprotein?

Kinase/Phosphataseswitch

GTPase switch

Regulationofproteinactivitybyakinase/phosphataseswitch.

• Cell-surfacereceptorsignaling– involveskinasephosphorylationandphosphatasedephosphorylation toregulatetargetproteinactivity.

• Proteinkinase– transfersterminalphosphatefromATPtospecificSer/Thr orTyr–OH(phosphorylatedresidueispartofaspecifickinasetargetmotif)

• Proteinphosphatase– hydrolyzesPoffproteinrestoringSer/Thr orTyr–OH

• Proteinkinasesandphosphatases–• Regulatedbysignalingprocesses• Modifyspecificproteintargetscontainingtargetmotifs

• Effectphosphorylation(andreversalbydephosphorylation)onproteinactivationordeactivation– protein-specific.

• Exampletargetprotein(reversedinotherproteins):

• unphosphorylated – inactive• phosphorylated– active

GTPaseswitch• GTPasesplayanimportantrolein:• Signaltransductionattheintracellulardomainoftransmembranereceptors,includingrecognitionoftaste,smellandlight.

• Proteinbiosynthesis(a.k.a.translation)attheribosome.

• Controlanddifferentiationduringcelldivision.• Translocationofproteins throughmembranes.• Transportofvesicleswithinthecell.(GTPasescontrolassemblyofvesiclecoats.)

GTPaseswitchproteinscyclebetweenactiveandinactiveforms.

• GTP-bindingproteins– signaltransductionpathwayon-offswitches• GTPaseproteinsuperfamily(GTP-binding,Gprotein):

• ON/active– GTPbound• OFFtoON–

• promotedbyGEFs(guaninenucleotideexchangefactors)• GEFscatalyzedissociationofboundGDPandreplacementbyGTP(notphosphorylationofGDP)

• OFF/inactive– boundGDP• ONtoOFF–

• GTPaseactivity– GTP→GDP+Pi(ONtoOFF)• AcceleratedbyGAPs(GTPase-activatingproteins)andRGSs(regulatorsofGproteinsignaling)

• GTPaseswitchproteins– twolargesignalingclasses:• Heterotrimeric– activatedbydirectinteractionwithsurfacereceptors(GEFs)• Monomeric– activatedbyGEFsthatareactivatedbysurfacereceptorsorotherproteins

What’stheswitch?Howdoesithaveafunciton?

Force

Change?What?

SwitchingmechanismofmonomericGproteins.

• GproteinON-OFFtransitionconformationalchanges:

• InvolveswitchIandswitchII(e.g.,RasmonomericGprotein)

• Promotesbindingtodownstreamsignalingproteins

• (a)Active/ONstate– boundGTP–

• (b)Inactive/OFFstate–boundGDP–

• IntrinsicGTPaseactivity– hydrolyzesGTPtoGDP(removesGTPγphosphate)

Ras signaling1695Analysis of Ras function in Drosophila

required for R8 differentiation are achieved by anotherreceptor system.

Different thresholds of Ras/MAPK activity inducedistinct cellular responses in the developing eyeThere are three different models for how specificity of Rassignaling is achieved: Specificity may be controlled by (1) thecellular context, (2) the activation of distinct signalingpathways by Ras or (3) by different levels of Ras activity. Theexperiments presented here support the importance of thecellular context and the different levels of Ras activity but failto provide evidence for the activation of different signalingpathways by Ras. All aspects of Ras signaling could berescued by the activation of the Ras/MAPK pathway and wefound no evidence, using the Ras effector site mutants, thatconstitutively active Ras activates the PI3K pathway directly.

The cellular context in which Ras activates MAP kinase isclearly important. Expression of RasG12V in blastoderm cellstriggers differentiation of head and tail structures (Melnick etal., 1993; Greenwood and Struhl, 1997), it triggers veindifferentiation in wing disc cells (Prober and Edgar, 2000) andneuronal differentiation in eye disc cells. We have shown herethat different levels of Ras/MAPK activity appear to controldistinct cellular responses within the same tissue (Fig. 7). Lowlevels of Ras activity, provided by the RasD38E mutant, rescueR8 differentiation and survival but not R1-R7 differentiation.High levels of Ras/MAPK activity provided by wild-type Rasor by a combination of RasD38E and rlSem are required for thedifferentiation of R1-R7 photoreceptor cells.

There are two possibilities with regard to the nature of theactivity thresholds that elicit the different cellular responses.The threshold may be quantitative. Cells could react todifferent activity levels within the cells. Alternatively, thethreshold may be temporal and cells react to the difference inthe duration of the signal. Staining of imaginal discs with anantibody that selectively recognizes activated MAPK (dpERK,Gabay et al., 1997) was not sensitive enough to detect activatedMAPK during normal photoreceptor cell recruitment or duringectopic neuronal differentiation in RasG12V-expressing clonesanterior to the morphogenetic furrow (data not shown).

Therefore, we cannot distinguish between these two models.In the present case, however, we favor the temporal modelbecause we do not detect the highest levels of dpERK stainingbehind the morphogenetic furrow during photoreceptor cellrecruitment. In response to MAP kinase activation in thedeveloping eye, a number of negative regulators of the pathwayare induced. The EGF-related peptide Argos competes with theTGFα-like ligand Spitz for EGF receptor binding (Freeman,1994; Golembo et al., 1996), and Sprouty, a cytoplasmicprotein, associates with the EGF receptor to turn off thesignaling pathway (Casci et al., 1999). Indeed, neuronaldifferentiation and ommatidial development in the RasD38E

mutant is rescued by the prolonged activity of MAPK causedby the rlSem mutation. It is possible that the reduced activity ofRasD38E towards Raf is caused by a more rapid inactivation,owing to increased GTPase activity. The observation thatRasG12V,D38E is sufficient to induce neuronal differentiationahead of the furrow, in conjunction with the G12V substitution,which inactivates the Ras GTPase activity is consistent withthe idea that D38E may stimulate GTP hydrolysis. Thus,neuronal differentiation in Drosophila may depend on theprolonged activation of Ras/MAP kinase, whereas transientactivation is sufficient for survival upon exit from the cell cycleand differentiation of R8 photoreceptor cells. Therefore itappears that neuronal differentiation in response to Rasactivation in the developing eye of Drosophila is similar toneuronal differentiation in PC12 cells, which also requiresprolonged activation of MAPK (Marshall, 1995). Themodulation of levels and/or the duration of Ras/MAPK activitylevels appear to be important determinants of cellularresponses in multicellular organisms.

We thank K. Basler, R. Böhni, B. Dickson, S. Leevers, M. Levineand S. Oldham for comments on the manuscript, and members ofthe Hafen and Basler laboratories for discussion. We thank J.Downward (ICRF) for sharing information about the effector sitemutations prior to publication. We also thank C. Berg, B. Dickson,B. Edgar and S. Leevers for fly stocks. This work was supported bythe Kanton of Zürich and Grants from the Swiss National Sciencefoundation and the Bundesamt für Bildung und Wissenschaft (EU-grants).

Ras RasD38E

Raf

rl/MAPK

Increase in clone sizeR8 differentiation

Postmitotic cell survival

C) Low Ras activity

Ras RasD38E

Raf

rlSem/MAPKSem

R1-R7 differentiationR1-R7 survival

D) Low Ras activity +

prolonged MAPK activity

Raf

rl/MAPK

RasG12V

Induction of precociousneuronal differentiation

E) High Ras activity

Ras

Raf

rl/MAPK

B) Loss of Ras function

Reduced growthapoptosis

Ras

Raf

rl/MAPK

A) Endogenous Ras activity

Cell growthPostmitotic cell survival

Photoreceptor differentiation

Fig. 7. Different thresholds of a single Ras effector pathway specify distinct cellular responses within the same cells. Our current model for Rasfunction in Drosophila eye development. (A) Ras is required for growth, cell survival and photoreceptor differentiation. These responses aremediated via Raf/MAP kinase signaling. (B) In the absence of Ras, mutant cells grow at a reduced rate, do not differentiate and die upon exitfrom the cell cycle. (C) Low levels or transient activation of MAPK achieved by RasD38E support cell survival and R8 differentiation.(D) Elevated levels or prolonged activation of MAPK achieved by combining RasD38E with the inactivation-resistant form of MAPK (RlSem)rescue R1-R7 photoreceptor differentiation and survival to the adult eye. (E) High levels or persistent activation by RasG12V or RasG12V, D38E individing cells lead to precocious photoreceptor differentiation.

1693Analysis of Ras function in Drosophila

or Raf activity is restricted to a zone anterior to themorphogenetic furrow. Clones in the antennal disc or in theperipodial membrane do not undergo neuronal differentiation.We conclude that in cells within the competence zone in theeye imaginal disc, high levels of Ras or Raf activity arenecessary and sufficient to induce neuronal differentiation. Inthe same cells, low levels of Ras activity promote growth. Theregulation of precise levels of Ras activity during eyedevelopment is therefore important for the correct cellularresponse.

DISCUSSION

We have presented evidence that Ras signaling controls

different cellular responses by at least two thresholds of MAPKactivity in the eye imaginal disc of Drosophila. Low levels ofMAPK activity permit growth, survival of postmitotic cells andR8 differentiation, while high activity induces R1-R7differentiation. These data significantly extend results fromprevious studies on the role of EGFR function during eyedevelopment. It provides direct evidence that, underphysiological conditions not involving overexpression, alteringthe levels of a single effector pathway, the Raf/MAPK pathway,is sufficient to elicit distinct cellular responses.

Ras and growth controlRas was first identified as an oncogene in vertebrates(reviewed by Bourne et al., 1990). Studies in tissue culturecells have suggested that the primary role of Ras is in thecontrol of cell proliferation, as the mitogenic response to avariety of growth factors can be blocked by inhibiting Rasfunction (Mulcahy et al., 1985; Smith et al., 1986). However,we show that in the eye imaginal disc, cells proliferate in theabsence of Ras, albeit with a reduced growth rate, implyingthat Ras is not essential for proliferation in this system. Thereason for the small size of Ras mutant clones compared totheir twin spots is not only the intrinsic growth deficit of thesecells but is caused by their failure to compete successfullywith the faster growing wild-type cells. These results are inagreement with a recent study of Ras function in the wingdisc (Prober and Edgar, 2000).

How does Ras control growth? One possibility is that Rasdirectly binds and activates PI3K. Clones mutant forcomponents in the insulin receptor/PI3K pathway also have agrowth disadvantage compared to wild-type cells (Böhni et al.,1999; Montagne et al., 1999; Weinkove et al., 1999). Althoughin vertebrates, H-RasG12V,Y40C activates PI3K, we found noevidence that the corresponding mutant activates PI3K inDrosophila. Partial loss-of-function mutations in genes codingfor Raf and MAPK, respectively, showed similar growthdefects as Ras mutants (Diaz-Benjumea and Hafen, 1994).Furthermore, RasD38E showed a significant rescue of thegrowth disadvantage of Ras−/− clones. Thus, we propose that

Fig. 5. Increasing MAP kinase activity permits RasD38E to rescue cellsurvival and photoreceptor differentiation. To test the rescue ofRasD38E in a background with increased MAPK activity weselectively removed Ras function in eye imaginal disc cells using theeyFLP, cell lethal technique (Newsome et al., 2000). The mutanttissue is marked by the absence of pigment. Genotypes: (A)eyFLP/+;FRT82B w+ cl3R3/TM3; (B) eyFLP/+; FRT82B Rasx7b/FRT82B w+ cl3R3; (C) eyFLP/+; P(Raswt,y+)/+; FRT82B Rasx7b/FRT82B w+ cl3R3; (D) eyFLP/+; P(RasD38E,y+)/+; FRT82B Rasx7b/FRT82B w+ cl3R3; (E) eyFLP/+; rlSem/+; FRT82B Rasx7b/ FRT82Bw+ cl3R3; (F,G) eyFLP/+; P(RasD38E,y+)/rlSem; FRT82B Rasx7b/FRT82B w+ cl3R3. eyFLP/+;FRT82B w+ cl3R3/TM3 flies showwild-type eyes (A). Selective removal of Ras function in the eye byeyFLP/+;FRT82B Rasx7b/FRT82B w+ cl3R3, generates flies with adrastically reduced head capsule and reduced eyes (B). One copy ofthe Ras rescue construct restores the head and the eye size (C). TheRasD38E transgene (D) or the rlSem mutation (E) alone do not rescuethe eye structure, but eye size is slightly increased due toundifferentiated cuticle in the eye (arrows). The RasD38E transgene ina rlSem background rescues Ras−/− photoreceptors (F). Tangentialsection through an eye shown in (F) reveals the presence ofdifferentiated R1-R7 photoreceptor cells (G).

Ras-Raf-MAPKpathway!

• RAS:RatSarcoma• Ras isa familyofrelatedproteins whichisexpressedinall animal celllineagesandorgans.AllRas proteinfamilymembersbelongtoaclassofproteincalled smallGTPase,andareinvolvedintransmittingsignalswithincells(cellular signaltransduction).


• RAFisanacronymfor Rapidly Accelerated Fibrosarcoma

• RAFkinases areafamilyofthree serine/threonine-specificproteinkinases thatarerelatedto retroviral oncogenes.Themousesarcomavirus3611containsaRAFkinase-relatedoncogenethatenhances fibrosarcoma induction.

• ActivationofRAFkinasesrequiresinteractionwith RAS-GTPases.

*A serine/threonine protein kinase (EC2.7.11.1)isa kinase enzyme that phosphorylates the OHgroup of serine or threonine


• Mitogen-activatedproteinkinases(MAPKs)areahighlyconservedfamilyofserine/threonineproteinkinases involvedinavarietyoffundamentalcellularprocessessuchasproliferation,differentiation,motility,stressresponse,apoptosis,andsurvival.

Ras-Raf-MAPKpathway

https://www.youtube.com/watch?v=r7GoZ9vFCY8

GainoffunctioninRas

Thinkwhythissignalingisnecessary

Secondmessenger?

Widespreadthesignal!

Secondmessenger• Secondmessengers aremoleculesthatrelaysignalsreceivedatreceptorsonthecellsurface— suchasthearrivalofproteinhormones,growthfactors,etc.— totargetmoleculesinthecytosoland/ornucleus.

Primarymessengers?

Fourcommonintracellularsecondmessengers.

• Intracellularsecondmessengerstransmitsignalsthroughthecytosol.

• cAMP:• GeneratedfromATPbyadenylylcyclase• ActivatesPKA

• cGMP:• Generatedbyguanylylcyclase• ActivatesPKGandspecificcationchannels

• IP3andDAG:• BothmadefromPIP2byphospholipaseC• IP3– openschannelstoreleaseCa2+ fromtheER

• DAG – withCa2+ activatesPKC• Calciumions(Ca2+) (notshown):

• Releasedfromintracellularstoresortransportedintothecell

• Activatescalmodulin,specifickinases(PKC),andotherregulatoryproteins

AsignaltransductionpathwayinvolvingaGprotein,asecondmessenger,aproteinkinase,andseveraltargetproteins

• Generalizedsignaltransductionpathway:• Step1:Hormonebindingtoitscell-surfacereceptor

• Step2:Activatedreceptor(GEF)activatestrimericGprotein

• Step3:Gproteinalphasubunitbindstoandactivatessecondmessenger-generatingenzyme.

• Step4:Activatedenzymegeneratesmultiplesecondmessengermolecules.

• Step5:Secondmessengeractivatesaproteinkinase.

• Step6:Kinasephosphorylatesandchangesactivityofoneormoretargetproteins.

• Step6a:Cytosolictargetproteinsinducechangesincellularfunction,metabolism,ormovement.

• Step6b:Targettranscriptionfactorsinducechangesingeneexpression.

Beta-adrenergicreceptor• The adrenergicreceptors (or adrenoceptors)areaclassof Gprotein-coupledreceptors thataretargetsofthe catecholamines,especially norepinephrine (noradrenaline)and epinephrine (adrenaline)

• Manycellspossessthesereceptors,andthebindingofacatecholaminetothereceptorwillgenerallystimulatethe sympatheticnervoussystem.Thesympatheticnervoussystemisresponsibleforthe fight-or-flight response,whichincludesdilatingthe pupils,increasingheartrate,mobilizingenergy,anddivertingbloodflowfromnon-essentialorgansto skeletalmuscle.

Gprotein• Gproteins,alsoknownas guaninenucleotide-bindingproteins,area

familyof proteins thatactas molecularswitches insidecells,andareinvolvedintransmittingsignalsfromavarietyofstimulioutsidea cell toitsinterior. Theiractivityisregulatedbyfactorsthatcontroltheirabilitytobindtoandhydrolyze guanosinetriphosphate (GTP)to guanosinediphosphate (GDP).WhentheyareboundtoGTP,theyare'on',and,whentheyareboundtoGDP,theyare'off'.Gproteinsbelongtothelargergroupofenzymescalled GTPases.

glycosylphosphatidylinositol-linked proteins (GPI)

AC• Adenylylcyclase (EC4.6.1.1,alsocommonlyknownas adenyl

cyclase and adenylatecyclase,abbreviated AC)isan enzyme withkeyregulatoryrolesinessentiallyall cells.

G-proteincoupledreceptor

https://www.youtube.com/watch?v=Nt2r5R0ZO5U


15.2StudyingCell-SurfaceReceptorsandSignalTransductionProteins• Near-maximalresponseofacelltoaparticularligandgenerallyoccursatligandconcentrationsatwhichlessthan100percentofitsreceptorsareboundtotheligand.

• Signalreceptorsandpathwaysaretargetedbynumerousdrugs.

• Receptorsandsignalingpathwayintermediatesarestudiedwithavarietyofexperimentalapproachesincludingaffinitychromatography,Westernblotting,immunoprecipitation,andpull-downassays.

Thinkthis!

Thinkthis!

Bindingassay

• Experimenttodeterminetheaffinityofareceptorforaligand:• Labeledligand– addedatvariousconcentrations(x axis)tocellsthatdo(experimentalcells)anddonot(controlcells)expressthereceptor

• Incubationinligand– 1hr (allowsbinding)at4°C(Lowtemperaturepreventsendocytosisofthecell-surfacereceptors.)

• Amountofboundligand(label)ismeasured (controlcelllabelbindingsubtractedfrombindingtoreceptor-expressingcells).

• Plot– boundligandpercellasafunctionoftheligandconcentration(red)

Bindingassay

• Results:• Atrelativelyhighligandconcentrations– numberofreceptor-boundligandmoleculesapproachesnumberoftotalcell-surfacereceptors

• Kd (halfmaximal)ligandbinding=1nM ligand• Parallelphysiologicalresponseexperimentsresults(blue):

• 18percentreceptorsboundtoligand– 50percentofthemaximalphysiologicalresponse• 50percentreceptorsboundtoligand– nearmaximalphysiologicalresponse

• Conclusion:relativephysiologicalresponseisgreaterthanligandbinding.

Isthestudyofreceptor-ligandbindingimportant?

Structuresofthenaturalhormoneepinephrine,thesyntheticagonistisoproterenol,andthesyntheticantagonistalprenolol.

• Syntheticanalogsofnaturalhormonesarewidelyusedbothinresearchoncell-surfacereceptorsandasdrugs– twoclasses:

• Maybindmuchmoretightlytothereceptorthandoesthenaturalhormone

• Maybemorestable• Agonist – mimicsfunctionofanaturalhormone–

• Bindstoandactivatesreceptor• Inducesthenormalcellularresponsetothehormone

• Antagonist – inhibitsfunctionofnaturalhormone• Bindstothereceptorligand-bindingsitebutinducesnoresponse

• Blocksnaturalhormonebinding• Reducesnormalphysiologicalactivityofthehormone

• Epinephrinereceptordrugs:• Structurallysimilartonaturalhormoneepinephrine• Isoproterenol– Agonist

• Binds~10xmorestrongly(10xlowerKd)thanepinephrine• Usedintreatingbronchialasthma,chronicbronchitis,andemphysema

• Alprenolol – Antagonist• Antagonistofcardiacmusclecellepinephrine-responsiveGprotein–coupledreceptor(β1-adrenergicreceptor)

• Receptoractivationincreasestheheartcontractionrate• Antagonists(beta-blockers)slowheartcontractions.• Usedintreatmentofcardiacarrhythmiasandangina

Proparanolol• Propranolol isa medication ofthe betablocker type. Itisusedto

treat highbloodpressure,anumberoftypesof irregularheartrate, thyrotoxicosis, capillaryhemangiomas, performanceanxiety,and essentialtremors

Agonistorantagonist?

Structure?

PTSDtreatment:PropranololisbeinginvestigatedasapotentialtreatmentforPTSD. Propranololworkstoinhibittheactionsof norepinephrine,a neurotransmitter thatenhances memoryconsolidation.

Nowyoucanunderstandthesignaling!!!

Nowyoucanthinkfortheprocess!

Howtostudythedownstreampathway?

• Bindingornot?• Phosphorylationornot?

Experiment

• Hematopoieticstemcell• PDGF• RacGTP

Hematopoietic stem cells (HSCs) or hemocytoblasts are the stem cells that give rise to all the other blood cells through the process of haematopoiesis.

Platelet-derivedgrowthfactor(PDGF)isoneofnumerousgrowthfactorsthatregulatecellgrowthanddivision.Inparticular,PDGFplaysasignificantroleinbloodvesselformation(angiogenesis),thegrowthofbloodvesselsfromalready-existingbloodvesseltissue.

Rac isasubfamilyoftheRhofamilyofGTPases,small(~21kDa)signalingGproteins(morespecificallyaGTPase).

Signaltransduction

1.BindingofRac toPAK?

2.Phosphorylation?

*p21-activated kinases (PAKs)

Pulldownassay

Thenlet’sstudythedownstreamofPAK!

What’sthePAK?Howtotestthephosphorylation?

Activationbythehormoneerythropoietin(Epo)ofthreesignaltransductionproteinsviatheirphosphorylation

• Westernblottingwithantibodyspecificforphosphorylatedsiterevealssignal-dependentphosphorylationoftargetproteins.

Thenlet’sstudythedownstreamofPAK!

NatureReviewsCancer 14, 13–25 (2014)Why?

Angiogenesis

HowmanyGPCRdothecellshave?


15.3GProtein–CoupledReceptors:StructureandMechanism• ThelargediversefamilyofGprotein-coupledreceptors(GPCRs) respondtoavarietyofextracellularsignalsandactivatetrimericGproteins.

• GproteinsfunctionasOn-Offswitchesforintracellularsignalingpathwaysbyactivatingorinactivatingionchannelsoreffectorenzymesthatgeneratesecondmessengermolecules.

• GPCRsignalingpathwaysregulateawiderangeofcellularactivitiesfrommetabolismtogeneexpression.

GeneralstructureofGprotein–coupledreceptors.

• Thesameorientationinthemembrane– N-terminusoutside,C-terminusincytosol

• Containseventransmembraneα-helicalregions(H1–H7)• Havefourextracellularsegments(E1–E4)• Fourcytosolicsegments(C1–C4)

• Ligandshavenotbeenidentifiedformany“orphan”receptors.

3DstructureofGPCR

Pocket?Conformationalchange?Whatinduces?

BindingofligandstoGPCRs

• (a)β1-adrenergicreceptorboundto antagonistcyanopindolol (ligand):

• H1-7– transmembranehelices• E2– oneof4extracellularloops• C2,C4– twooffourcytosolicdomains

• (b)Hormone-bindingpocket:• Sidechainsof15aminoacidsinfourtransmembraneαhelices(3,5,6,and7)andoneextracellularloopE2makenoncovalentcontactswiththeligand.

• Examplesofspecificbindinginteractions:Natominbothincyanopindolol andinepinephrineformsanionicbondwiththecarboxylatesidechainsofhelix3aspartate121(D)andhelix7asparagine329(N).

• (c)Glucagon(29-amino-acidpeptide)bindingtotheglucagonreceptor:

• GlucagonC-terminus(red)– bindstothereceptorN-terminaldomain

• GlucagonN-terminus– thoughttoinsertintoabindingpocketthatisinthecenteroftheseventransmembraneαhelices

GeneralmechanismoftheactivationofeffectorproteinsassociatedwithGprotein–coupledreceptors

• Ligand-activatedGprotein–coupledreceptors– (GEFs)catalyzeexchangeofGTPforGDPontheαsubunitofaheterotrimericGprotein

• Box– trimericGproteinGα andGβγsubunits– tetheredtothemembranebycovalentlyattachedlipidmolecules(wigglyblacklines)

• Step1:Ligandbindinginducesreceptoractivationconformationalchange.

• Step2:ActivatedreceptorbindstotrimericGprotein.

• Step3:ActivatedreceptorGEFactivitystimulatesGα subunitreleaseofGDP.

• Step4:GTPbindingchangesGαconformation –

• DissociatesGβγ (Gβγ subunitactivatesothereffectorenzymesinsomepathways.)

• ActivatesGα• Step5:Gα·GTPactivateseffectorenzyme.• Step6:Gα intrinsicGTPase activity

hydrolyzesGTPtoGDP– dissociatesGαandturnsoffeffectorenzyme.(Gproteinactiveforminutesorless)

HowtoshowtheactivationofG-proteinactivationinthecell?

ActivationofaGproteinoccurswithinsecondsofligandbindingtoitscell-surfaceGprotein–coupledreceptor

• Forsterresonanceenergytransfer(FRET)technique:

• Gα-cyanfluorescentprotein(CFP,excitedby440-nmlight,fluoresces490-nmlight)• Gβ-yellowfluorescentprotein(YFP,excitedby490-nmlightemittedfromCFP,fluoresces527-nmlight)

• (a,left)InactiveGα·Gβγ complex– CFPandYFPcloseenoughforenergytransfer• ExcitationofCFPwith440-nmlight–

• causesfluorescenceenergytransferfromactivatedCFPtoYFP• emissionof527-nm(yellow)lightinsteadof490-nm(cyan)light

• (a,right)activatedG-protein– dissociationoftheGα andGβγ subunits– CFPandYFPnotcloseenoughforenergytransfer• ExcitationofCFPwith440-nmlight• Lossof527-nmlightemission– emissionof490-nm(cyan)lightinstead

• (b)Results:plotofyellowlight(527nm)emissionfromasingletransfectedamoebacellbeforeandafteradditionofextracellularcAMP(arrow)

• Conclusion:Extracellularsignal-GPCRinteractionstimulatesGproteinactivationwithinseconds.

Structureoftheβ2-adrenergicreceptorintheinactiveandactivestatesandwithitsassociatedheterotrimericGprotein,Gs

• Ligandbindingconvertsβ2-adrenergicreceptorintoaconformationthatbindsitstrimericGprotein.

GPCR

• RobertLefkowitzandBrianKobilka – awarded2012NobelPrizeinChemistryforworkonβ2-adrenergicreceptor,includingstructuredetermination

MajorClassesofMammalianHeterotrimericGPorteinsandtheirEffectors

GPCR

https://www.youtube.com/watch?v=Glu_T6DQuLU


15.4GProtein–CoupledReceptorsThatRegulateIonChannels• ThecardiacmuscarinicacetylcholineGPCRregulatesaK+channel.

• LightstimulationofthephotosensitiverhodopsinGPCRclosescGMP-gatedNa+/Ca2+ channelsbyregulatingacGMPpathwayinretinalcells.

• Severalmechanismsacttoterminatevisualsignaling.• AdaptationtoawiderangeofambientlightlevelsismediatedbymovementsoftheGproteintransducinandtheinhibitorproteinarrestinintoandoutoftherod-celloutersegment.

Inheartmuscle,themuscarinicacetylcholinereceptoractivatesitseffectorK+ channelviatheGβγ subunitofaGi protein

• Gβγ subunit(ratherthanGαi-GTP)bindstoandopensaK+ channel(effectorprotein).

• IncreasedK+ exithyperpolarizesthecardiacmusclecellmembrane– reducesheartmusclecontractionfrequency

Rodcell

Rodcell

• Rodcells are photoreceptorcells inthe retina ofthe eye thatcanfunctioninlessintense light thantheothertypeofvisualphotoreceptor, conecells.Rodsareusuallyfoundconcentratedattheouteredgesoftheretina andareusedin peripheralvision.

Conecell• Conecells,or cones,areoneofthreetypesof photoreceptorcells in

the retina of mammalian eyes(e.g.the humaneye).Theyareresponsiblefor colorvision andfunctionbestinrelativelybright light,asopposedto rodcells,whichworkbetterindimlight.Conecellsaredenselypackedinthe foveacentralis,a0.3 mmdiameterrod-freeareawithverythin,denselypackedconeswhichquicklyreduceinnumbertowardstheperipheryoftheretina.Thereareaboutsixtosevenmillionconesinahumaneyeandaremostconcentratedtowardsthe macula.

Humanrodcell.

• RhodopsinGPCRsenseslightinrodcells.

• (a)Rodcellschematicdiagram:• Rhodopsin– locatedintheflattenedmembranedisksofthecelloutersegment

• Synapticbody– synapseswithoneormoreinterneurons

• (b)EMoftheregionoftherodcellbracketedregionin(a)–junctionoftheinnerandoutersegments

Visiondependsonthelight-triggeredisomerizationoftheretinalmoietyofrhodopsin.

• Rhodopsin:• Activatedbyabsorbingenergyfromphotonoflight

• Opsinproteinlysine296aminogroup– covalentlyattachedtothelight-absorbingpigment11-cis-retinal

• Lightabsorption:• Causesrapidphotoisomerization oftheboundcis-retinaltotheall-transisomer

• Triggersrhodopsinconformationalchangetotheactivatedunstableintermediatemeta-rhodopsinI

• ActivatedrhodopsinactivatesGtprotein(transducin)

Transducin

• Transducin (Gt)isaproteinnaturallyexpressedinvertebrateretinarodsandconesanditisveryimportantinvertebratephototransduction.ItisatypeofheterotrimericG-proteinwithdifferentαsubunitsinrodandconephotoreceptors.

Rhodopsin

Transducin

Thelight-activatedrhodopsinpathwayandtheclosingofcationchannelsinrodcells.

• Dark-adaptedrodcells:• HighlevelofcGMP– keepscGMP-gatednonselectivecationchannelsopen• Openchannelsdepolarizetheplasmamembraneto~−30mV,considerablymorepositive(less

negative)thanrestingpotential(−60to−90mV)typicalofneuronsandotherelectricallyactivecells.• Stimulatesneurotransmitterrelease

Membranepotential?

t

Lighton

-30mV

Membranepotential?

t

LightonHowtogoback?

-30mV

PDEinactive

Inhibitionofrhodopsinsignalingbyrhodopsinkinase.

• Processofrhodopsinphosphorylationandinactivationbyarrestin – completedveryquickly,within50milliseconds

• Feedbackrepressionofoveractivated rhodopsin

Nowyoucanimagineyourowncellsignaling…

Rhodopsin

Transducin

Arrestin

Dark- andlight-adaptedrodcells

• Sensitivityandproteinlocation

• Darkadaptation?• Lightadaptation?

• Transducin andArrestin location?

Schematicillustrationoftransducin andarrestin distributionindark-adaptedandlight-adaptedrodcells.

• (a)Dark(visionmostsensitivetoverylowlightlevels)

• Transducin – mostlocalizedinoutersegmentmembranes

• Arrestin – mostlocalizedininnersegmentregionofthecell

• (b)Brightlight(visionisrelativelyinsensitivetosmallchangesinlight):

• Transducin – movedfromoutersegmenttoinnersegment

• Arrestin – movedfrominnersegmenttooutersegment

• Coordinatedmovementoftransducin andarrestin:• Contributestoabilitytoperceiveimagesovera100,000-foldrangeofambientlightlevels

• Proteinmovementmechanism– mayinvolvemicrotubulesandmotorproteins

Visualsignaling

https://www.youtube.com/watch?v=JIPE3in2EcQ


15.5GProtein–CoupledReceptorsThatActivateorInhibitAdenylylCyclase• GPCRsactivateGproteinsthatactivateorinhibitadenylylcyclasegenerationofcAMPfromATPandareregulatedbyfeedbackrepression.

• cAMPactivatesproteinkinaseA(PKA),whichphosphorylates-regulatesmultipletargetproteinsincludingenzymesincells.

• EpinephrineactivationofitsGPCRinliverandmusclecellsstimulatesglycogenbreakdownintoglucosebyinhibitingglycogensynthesisandstimulatingglycogenbreakdownviaakinasecascade.

• PKAactivationcanstimulategeneexpression.

SynthesisandhydrolysisofcAMP byadenylylcyclaseandPDE.

•Adenylylcyclase(AC)–catalyzesformationofcycliccAMP (secondmessenger)bondfromATPprecursor

• cAMP phosphodiesterase(PDE) – catalyzeshydrolysisofcyclicbond– AMP(notsecondmessenger)

Hormone-inducedactivationandinhibitionofadenylylcyclaseinadiposecells.

ActivationofthecatalyticdomainofmammalianadenylylcyclasebybindingtoGαs·GTP.

• (a)Mammalianadenylylcyclase(AC)–membrane-boundenzyme:

• Twosimilarcatalyticdomains– eachconvertATPtocAMP onthecytosolicfaceofthemembrane

• Twointegralmembranedomains– eachcontainssixtransmembraneαhelices

StructureofPKAanditsactivationbycAMP

• (a)PKA:• Twocatalytic(C)kinasesubunits–transferterminalphosphatefromATPtotargetproteinspecificSer/Thr-OH

• Tworegulatory(R)subunits• (-)cAMP – bindandinhibitcatalyticsubunitphosphorylationactivity

• (+)cAMP – releaseactivecatalyticsubunits

GPCR&cAMP

https://www.youtube.com/watch?v=0nA2xhNiAow

AmplificationofanextracellularsignalbyasignaltransductionpathwayinvolvingcAMP andPKA.

ActivationofCREBtranscriptionfactor followingligandbindingtoGαs-coupledGPCRs

• cAMP-PKAregulatesgeneexpressionthroughCREB.

• Step1:ReceptorstimulationleadstoriseincAMP.

• Step2:cAMP activatesPKA.• Step3:PKAcatalyticsubunitstranslocateintothenucleus.

• Step4:PKAphosphorylates-activatestheCREBtranscriptionfactor.

• Step5:• ActivatedCREBformscomplexwiththeco-activatorCBP/P300andotherproteins.

• CREBcomplexbindstoCREregulatoryelementsinpromotersofmultiplegenes.

• CREBcomplexbindingstimulatestranscriptionofthevarioustargetgenescontrolledbyaCRE.


15.6GProtein–CoupledReceptorsThatTriggerElevationsinCytosolicandMitochondrialCalcium• GPCR-GproteinactivationofphospholipaseC

generatesIP3 (soluble)andDAG(membranebound)secondmessengersfromPIP2.

• IP3 triggerstheopeningofIP3-gatedCa2+ channelsintheendoplasmicreticulumandelevationofcytosolicfreeCa2+,whichactivatesPKCandcalmodulin.

• NeuralandhormonalstimulationcoordinatelyregulateglycogenbreakdownthroughCa2+ andcAMP.

• AcetylcholineactivationofitsGPCRonendothelialcellsinducesgenerationoftheNOgaseoussignal,whichstimulatessmoothmusclerelaxationandvasodilation.

SynthesisofsecondmessengersDAGandIP3 fromphosphatidylinositol(PI).

TheIP/DAGpathwayandtheelevationofcytosolicCa2+.

• (a)OpeningofendoplasmicreticulumCa2+channels:

• Step1:GPCRactivationofeithertheGαoorGαq subunit– activatesphospholipaseC(PLC).

• Step2:PLCcleavesPI(4,5)P2– yieldsIP3 andDAG

• Step3:IP3 diffusesthroughthecytosol– IP3interactswithandopensIP3-gatedCa2+channelsintheERmembrane

• Step4:Ca2+ ionsmovedownconcentrationgradientthroughthechannelintothecytosol.

• Step5:Ca2+ bindingactivatesPKCanditsrecruitmenttotheplasmamembrane.

• Step6:DAGactivatesmembrane-associatedPKC.

• Step7:ActivatedPKC-Ca2+ leavesmembranetophosphorylatevariouscellularenzymesandtranscriptionfactors,activatingproteinsinvolvedincellgrowthandmetabolism.

What’stheadvantageofDAG?

OscillationsinthecytosolicCa2+ concentrationfollowingtreatmentofhumanHeLacellswithhistamine.

• HighcytosolicCa2+ level>10-4 MCa2+:• DecreasesCa2+ channelaffinityforIP3 –channelsclose(Ca2+ – feedbackinhibitorofIP3-gatedCa2+ channels)

• InhibitsfurtherIP3-inducedreleaseofCa2+ fromERstore,eveninelevatedIP3

• Ca2+ ATPasepumpsCa2+ backintoER– lowerscytosolicCa2+ concentration

• LowcytosolicCa2+ concentration–potentiatesIP3 reopeningofIP3-gatedCa2+channelsifsignalpersists

Whathappens?

Integratedregulationofglycogenolysis byCa2+ andcAMP/PKApathways

Glycogenbreakdown


• Neuronalstimulation–• CytosolicCa2+ increase• Ca2+ bindstoandactivatesglycogenphosphorylasekinase(GPK)– increasesglycogenbreakdown

• Epinephrinebindingtoβ-adrenergicreceptors –

• cAMP increase• cAMP activatesPKA• PKAphosphorylates-activatesGPK – increasesglycogenbreakdown

• PKAphosphorylates-inhibitsglycogensynthase(GS)– inhibitsglycogensynthesis


• Hormonalstimulationoftwoβ-adrenergicreceptorpathways–

• Epinephrine– cAMP increase–activationofPKA

• PKA–• Phosphorylates-activatesGPK–increasesglycogenbreakdown

• Phosphorylates-inhibitsGS–inhibitsglycogensynthesis

• Vasopressin –• IP3 increase– cytosolicCa2+increase

• Ca2+ bindstoandactivatesglycogenphosphorylasekinase(GPK)– increasesglycogenbreakdown

• enhancesPKCactivationbyDAG

• DAGincrease– activationofPKC• PKC– phosphorylates-inhibitsGS– inhibitsglycogensynthesis

Differentpathways=>cAMP andCalciumregulations!

TheCa2+/nitricoxide(NO)/cGMPpathwayandtherelaxationofvascularsmoothmuscle

• Endothelialcellssignalingtosmoothmusclecells:

• AcetylcholineactivationofitsGPCR–

• Step1:G-proteinactivationofPLC• Step2:PLCgenerationofIP3 (+DAG)– cytosolicCa2+ increaseactivatescalmodulin

• Step3:Ca2+-calmodulinactivatesNOsynthase

• Step4:NOsynthase– generatesNO(nitricoxide,gaseoussignalmolecule)

• Step5:NOdiffuseslocallyintosmoothmusclecells– activatesguanylylcyclase(NOreceptor)

• Step6:GuanylylcyclasegeneratescGMP(secondmessenger)

• Step7:cGMPactivatesproteinkinaseG(PKG).

• PKGphosphorylatestargetproteins– decreasescytosolicCa2+ – smoothmusclerelaxation

Discussionwithfriends

www.abcam.com

Ras Signaling

1.HereistheRas signaling.PleasefindRas-Raf-MAPKpathwayandexplaintheresult.

2.FindoneexampledrugwhichregulatestheGPCRsignaling.

Neurobiology of Learning and Memory 74, 259–266 (2000)doi:10.1006/nlme.1999.3950, available online at http://www.idealibrary.com on

Impaired Memory Consolidation in RatsProduced with !-Adrenergic Blockade

Larry Cahill,* Christian A. Pham,* and Barry Setlow†

*Department of Neurobiology and Behavior, and Center for the Neurobiology of Learning and Memory,University of California, Irvine, California 92697-3800; and †Department of Psychology,

Johns Hopkins University, Ames Hall, 3400 North Charles Street, Baltimore, Maryland 21218

Despite abundant evidence that systemic administration of adrenergic drugs andhormones can produce retrograde memory enhancement, the literature containsno clear demonstration that postlearning systemic administration of adrenergicantagonists produces retrograde amnesia. Here we demonstrate retrograde amnesiafor a stressful learning task (a spatial water maze) with systemic administration ofthe !-adrenergic antagonist propranolol (5 mg/kg). The amnesic effect of the drugdepended on the degree of learning in the subjects: Propranolol caused a robustretrograde amnesia in “good learners,” but did not significantly affect memory in“poor learners.” The findings provide critical additional support for the hypothesisthat postlearning adrenergic activation modulates memory consolidation processesafter emotionally stressful events and help explain previous failures to detect mem-ory impairment after systemic administration of adrenergic blocking drugs.! 2000 Academic Press

On the basis of early evidence for the retrograde enhancing effects of drugs on memorystorage processes (e.g., Breen & McGaugh, 1961), Gerard (1961) suggested that epineph-rine released by a “vivid emotional experience” may influence the “fixation process”of memory. Since that suggestion, substantial evidence has been generated implicatingadrenergic mechanisms in memory consolidation. Retrograde memory enhancement withsystemically administered adrenergic agonists has been found in many experimental tasksand species, including humans (McGaugh et al., 1984, 1996; Soetens et al., 1995). Themost widespread interpretation of these well-established retrograde effects is that theadrenergic system is involved with memory consolidation, in particular for emotionallyarousing learning experiences that induce adrenergic hormone release (McGaugh et al.,1984, 1996).Despite the abundant evidence for adrenergic participation in memory consolidation

and clear evidence of the central administration of adrenergic agents influencing memory(McGaugh et al., 1984, 1996; Roullet & Sara, 1998; Sara et al,. 1999) there are no cleardemonstrations of retrograde memory impairment produced by systemically administered

Address correspondence and reprint requests to Larry Cahill, CNLM, UC Irvine, California 92697-3800. Fax:(949) 824-5244. E-mail: [email protected].

259 1074-7427/00 $35.00Copyright ! 2000 by Academic Press

All rights of reproduction in any form reserved.

3.Findthispaperandexplainthefigure1withthebeta-adrenergicpathway.

4.FindthemechanismofViagraanditssideeffectsintermsofthemolecularsignaling.

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