The cell biology of neuronal synapsesMorgan Sheng617.452.3716([email protected])The Picower Center for Learning and MemoryNumerousVariable in sizeHeterogeneous in compositionDynamicSheng M, Kim MJ. (2002) Postsynaptic Signaling and Plasticity Mechanisms. Science. 298:776-780.PSDSVCentral excitatory synapse (asymmetric, glutamatergic) AMPArNMDArScale bar = 20 nm40 nm40 nmEM image of native tetrameric AMPA receptors purified from brainWhat is the resolution of EM? What is the size of AMPA receptor?Glutamate receptor-channels mediateexcitatory synaptictransmissionNMDA subtypeAMPA subtypeKainate subtypeWhat’s the affinity of GluRs for glutamate?Toyoshima C and Unwin N. 1990. Three-dimensional structure of the nicotinicacetylcholine receptor from Torpedo electric organ by cryoelectron microscopy.J Cell Biol. 111:2623-35.The most famous postsynaptic membrane is the muscle end-plateDensity of AChR in muscle postsynapticmembrane is ~10,000 per sq um7TM4TM "ligand-gated channel superfamily"nAChRGlyRGABA(A)R5HT3R3TM plus "P-loop" Glutamate receptorsNMDA / AMPAR / KAR/ Delta2TM plus "P-loop"P2X (ATP) receptorheptahelicalserpentineGPCR (G protein)frizzled, smoothenedMembrane topology of ligand-gated ion channels (or ionotropic receptors)G-protein coupled receptorsNCNCNCNCAMPAr: GluR1-4NMDAr: NR1, NR2A-DHow were these receptors cloned?C. elegans Glr-1-GFP in ventral nerve cord (Rongo and Kaplan 1999)DGluRIIB at fly NMJ.Top, HRP immunocytochemistryBottom, immunofluorescence: Green,Myc-GluRIIB; Red, synaptotagmin(Peterson et al 1997 [Goodman lab])Colocalization of PSD-95 with AMPAR-GluR1 in cultured hippocampal neurons(Rao A et al 1998 [Ann Marie Craig])Glutamate receptors are specifically targeted to excitatory synapsesExcitatory synapses red, inhibitory synapses blueKristen Harris and colleaguesUsually one excitatory synapse per spine, on spine headmGluRNMDAAMPAHow are glutamate receptors targeted and clustered in the postsynaptic membrane? How are they segregated from inhibitory (GABA) receptors?How are they aligned with the correct axon terminals?What kinds of experimental approach?ImpuritiesCloning requires peptide seq (butnow mass spec);synapses heterogeneous; receptorsnot abundant (except TorpedoE.O.)Informs about NATIVE complexStraightforward,Informs about size, biochemicalpropertiesPurification ofreceptor complexfrom tissueFalse positivesRequires known cloned protein asbait (affinity ligand), but Y2Hdoes not need purification ofligand;Can only isolate direct bimolecularinteractors;Informs about direct protein-proteininteraction;Amenable to high thruput screening;Does not require solubility of ligand orinteractorYeast two-hybridscreen for receptorbinding proteinsNeed purified affinity ligand (iecloned receptor)Interacting protein needs to beeasily solubilized and activeInforms about protein-protein interaction;affin purification can work for ternarycomplex, indirect interactionAffinity purificationof receptor bindingproteins eg via GSTfusionScreening labor intensive;Has to be done in “genetic”organism;Redundancy, lethalityGives you “function” at cellular andorganismal level; Not biased byabundance or biochemical propertiesForward genetics:mutants thatmislocalize receptorapproach advantage disadvantagemGluRNMDAAMPAPostsynaptic glutamate receptors interact with distinctscaffolding / anchoring proteinsPSD-95GRIPPDZPDZdomaindomain(D. Bredt, C. Garner, S. Grant, R. Huganir, M. Kennedy,P. Seeburg, P. Worley, E. Ziff)IESDVESVKINMDAR2/NR2GluR2PDZ2 ofPSD-95PDZ5 of GRIPDifferential specificityOf PDZ domains(recognize ~4 aa at C-term)(Class 1)(Class 2)25Arg 318NCβB2βC4αAβAβCβEαB-2-1-3Carboxylate binding loopNCβB5βB2βAβBβDαB0βFαB1Structural basis PDZ domain – C-terminal bindingPDZ domains recognize at least 4 C-terminal residues0, -2 positions most important for specificityRecognition of the terminal carboxylate by a conserved loopPDZ domain proteinsPresent in bacteria to mammalsAbundantly represented in sequenced genomes (100s)Typically contain multiple domains : “scaffold” proteinsBind to a specific set of proteins –organize a specific signaling complexAssociated with specific domains of membranee.g. epithelial cell tight junctions, synapsesCan be relatively fixed in location (anchoring proteins)or relatively dynamic (involved in trafficking)The “logic” of C-terminal targeting motifsThe variety ofproteins containingPDZ domains andother protein-proteininteraction modulesSH3 GuKPKCPKCPKCCaMCaMPKCrhodopsinrhodopsinGαqTRPTRPCharles Zuker,Craig Montell etcInaD, a protein with 5 PDZ domains that organizes thephototransduction cascade in DrosophilamGluRNMDAAMPAPostsynaptic glutamate receptors interact with distinct scaffolding proteins and assemble into different signaling complexesPSD-95HomerGRIPPDZPDZdomaindomain(D. Bredt, C. Garner, S. Grant, R. Huganir, M. Kennedy,P. Seeburg, P. Worley, E. Ziff)nNOSPSD-95nNOSNMDARExample of PSD-95 mediated “functional complex” of NMDAR and nNOSnNOSiscalcium/calmodulinregulatedenzymeSpecificallycoupledtocalciumentrythroughNMDAreceptorsBindsspecificallytoPDZ2ofPSD-95(PSD-95alsobindstoNMDAreceptorsviaPDZ1andPDZ2).PSD-95isbelievedtophysicallylinkandfunctionallycouplenNOStoNMDAreceptor.How would you test that thiscomplex exists and that it isimportant for NMDAR-nNOS coupling?Antisense knockdown of PSD-95 reduces NO productioninduced by NMDA receptor activationInhibition of PDZ2 interactions of PSD-95 inhibits nNOSactivation by NMDA receptorsand reduces excitotoxicity following ischemia/stroke in cultureand in rodentsFunctional coupling by PSD-95 scaffold: example of nNOSNMDA receptor function is unaffected by aboveHomosynaptic hippocampal CA1 LTP and LTD0.511.522.50 10 20 30 40 5000.511.50 10 20 30 40 501 Hz, 900 pulses100 Hz, 100 pulsesTime (min) Time (min)100 Hz, 100 pulses  LTP1 Hz, 900 pulses  LTDAMPA receptorConstitutivecycling ofAMPA-Rsbetweensurface andintracellularpoolssurfaceintracellularSynaptic plasticity can be mediated by presynaptic or postsynapticchangesAMPA receptorStrengthensynapse bydelivery of moreAMPARs tosurface (dependson GluR1)Surface IntracellularAMPA receptorWeaken synapseby removingAMPARs fromsurface (dependson GluR2)Surface IntracellularLysosomedegradationLTP requires exocytosis/delivery of AMPA receptors tothe postsynaptic membrane(depends on GluR1 subunit)LTD requires