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8/9/18
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IntroductiontoBone:physiologyanddiseaseAmini-coursedesignedforsummerstudents;SponsoredbytheCenterforSkeletalResearch
Thursday,July19,2018
Cell Signaling
Tom Gardella, PhD
Stimulus(ligand)
ReceptorSecondMessengers
Effectors
CellSignalingLigands arestimulithatbindtospecificreceptors.Hydrophilicligandsbindreceptorsonthecellsurface.Hydrophobicligandsbindreceptorsinsidethecell.
Down-streamresponsesincludeeffectsonothercellsurfaceproteins(e.g.channelsandtransporters);changesincellshapeormotility,andexpressionandsecretionofotherstimuli,suchashormones,cytokinesandcell-cellinteractionproteins.
Effectors areintermediaryproteinsthatrelayinformationinthecell;effectorsmayamplifythesignalbyproducingsecondmessengermolecules,suchascAMP.Effectorsandsecondmessengersformsignalingcascades.
Signalingisregulated topreventexcessstimulation.
ReceptorConformationalChangeReceptorstransduceinformationbychangingconformation.Ligandsbindtoaninactivereceptorconformationandinduceanactive-stateconformation,which cancoupletoeffectorproteins.
inactive active
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LigandPharmacologyIa – ligandtypes
Agonistligandsbindreceptorandinduceanactive-stateconformationthatcancoupletoeffectors.
Antagonistsareligandsthatbindandinduceaninactiveconformationthatcannotcoupletoeffectors;suchligandsactasreceptorblockers.
Inverseagonistsareligandsthatbindtoactive-statereceptorsandinducetheinactiveconformation;suchligandslowerthebasalsignalinglevel.
Partialagonistsinducelessthanthemaximumresponseinducedbyafullagonist.
Agonist
InverseAgonist
Antagonist
PartialAgonist
LigandPharmacologyIb - potencyandefficacyPotency– Theligand(ordrug)concentrationthatinduceshalfofthemaximumresponse,expressedasEC50 (nMorLogM)orpEC50(-logM).
Efficacy– Themaximumresponseinducedbyaligandordrug(Emax,expressedtypicallyasmolesofproductpercellorwell,oras%).
N.B.Potencyandefficacyofadrugorligandaredependentonthesystem–i.e.thecelltype,thenumberofreceptors,effectors,etc.).Apartialagonistinonesettingmaybeafullagonistinanother.
EC50
Emax
LigandPharmacologyIc biasedagonism
Agonist1
Receptorsarepleiotropicandcanadoptdifferentconformationsinresponsetostructurallydistinctligandsandthusactivatedifferenteffectorpathways– biasedagonism.
Agonist2
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CellMembraneReceptors
GProtein-CoupledReceptors
ReceptorTyrosineKinases
GPCRs• ~800GPCRsinhumans(~3%ofproteome)•Largestintegralmembraneproteinsuperfamily•Mediateresponsestodiversestimuli:peptidehormones,neurotransmitters,cytokines,
mineralions,aromas,andphotons.•Targetedby30-50%ofalldrugs.•Fourmaingroups:
Group #A 287 (adrenergic receptors, rhodopsin) B 16 (peptide hormone receptors- PTHR1)C 22 (m-glutamate and CaSR)F 11 (Frizzled-wnts) olfactory (A) 417adhesion (B2) 33 other 49
GPCR-GProteinCoupling
AgonistbindinginducescouplingtoaheterotrimericGprotein comprisedofabgsubunits.
CouplinginducesGDPàGTPexhangeinGa andtrimer dissociation.
Ga-GTP(andsometimesGb/g) activatedown-streameffectorsandsecondmessengers.
InactivationoccursuponGTPhydrolysis toGDP(enhancedbyRGS-GAPs)andtrimer reassembly.
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RhoA
GProteinCascades
Gas Ga
12/13AC
cAMP
PKA
Gai
AC
cAMP
Gaq/11
PLC
IP3
Ca++ PKC
+DAGROCK
DifferentGPCRscoupletodifferentGproteins,whichactivatedifferenteffectorcascades.
Zhang et al. Nature 2018
GlucagonRecptor
Mechanism of Receptor Activation Agonist binding to the extracellular surface causes the cytoplasmic surface to open.
Receptor-G Protein Interaction
Helix-5 of Ga docks into the cytoplasmic cavity of the GPCR.
Docking induces a movement of helix-5 that causes release of GDP and G protein activation.
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SignalTermination
ActivatedGPCRsarephosphorylatedontheC-tailbyGproteinreceptorkinases(GRKs).
Thephosphorylatedreceptorrecruitsarrestin.
Arrestin recruitsAP-2andclatherin, whichassemblesintocoatedpits.
Clatherin-coatedpits budfromthemembraneandmoveintothecytoplasmtobecomeendosomes.
Endosomestrafficthroughthecellandmediatereceptordegradationorrecyclingtothecellsurface.
Signalingusuallyterminateswitharrestin binding,butmaycontinueinendosomes,dependingontheligand.
AllostericModulation
Receptoractivitycanbemodulatedbyallosteric drugs,whichbindoutsideoftheorthosteric siteusedbythenaturalligand.
PAMs,orPositiveAllostericModulators,increaseactivityand/orresponsetoorthosteric agonistligands.
NAMs,orNegativeAllostericModulators,decreaseactivityand/orresponsetoorthosteric agonistligands.