8/9/18

1

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

8/9/18

2

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

8/9/18

3

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.

8/9/18

4

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.

8/9/18

5

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.