RESUMO
The postsynaptic density protein PSD-95 influences synaptic AMPA receptor (AMPAR) content and may play a critical role in LTD. Here we demonstrate that the effects of PSD-95 on AMPAR-mediated synaptic responses and LTD can be dissociated. Our findings suggest that N-terminal-domain-mediated dimerization is important for PSD-95's effect on basal synaptic AMPAR function, whereas the C-terminal SH(3)-GK domains are also necessary for localizing PSD-95 to synapses. We identify PSD-95 point mutants (Q15A, E17R) that maintain PSD-95's influence on basal AMPAR synaptic responses yet block LTD. These point mutants increase the proteolysis of PSD-95 within its N-terminal domain, resulting in a C-terminal fragment that functions as a dominant negative likely by scavenging critical signaling proteins required for LTD. Thus, the C-terminal portion of PSD-95 serves a dual function. It is required to localize PSD-95 at synapses and as a scaffold for signaling proteins that are required for LTD.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Proteínas de Membrana/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Espinhas Dendríticas/fisiologia , Proteína 4 Homóloga a Disks-Large , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Modelos Biológicos , Neurônios/ultraestrutura , Técnicas de Patch-Clamp/métodos , Mutação Puntual/fisiologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/fisiologia , Transmissão Sináptica/fisiologia , Transmissão Sináptica/efeitos da radiação , Transfecção/métodosRESUMO
The postsynaptic density (PSD) consists of a lattice-like array of interacting proteins that organizes and stabilizes receptors, ion channels, structural, and signaling proteins necessary for synaptic function. To study the stabilization of proteins within this structure and the contribution of these proteins to the integrity of the PSD, we tagged synaptic proteins with PAGFP (photoactivatable green fluorescent protein) and used combined two-photon laser-scanning microscopy and two-photon laser photoactivation to measure their rate of turnover in individual spines of rat CA1 pyramidal neurons. We find that PSD-95 is highly stable within the spine, more so than other PSD-associated proteins such as CaMKIIalpha, CaMKIIbeta, GluR2, and Stargazin. Analysis of a series of PSD-95 mutants revealed that distinct domains stabilize PSD-95 within the PSD and contribute to PSD formation. Stabilization of PSD-95 within the PSD requires N-terminal palmitoylation and protein interactions mediated by the first and second PDZ domains, whereas formation of a stable lattice of PSD-95 molecules within the PSD additionally requires the C-terminal SH3 domain. Furthermore, in a PDZ domain 1 and 2 dependent manner, activation of NMDA receptors with a chemical long-term depression protocol rapidly destabilizes PSD-95 and causes a subset of the PSD-95 molecules previously anchored in the spine to be released. Thus, through the analysis of rates of exchange of synaptic PSD-95, we determine separate domains of PSD-95 that play specific roles in establishing a stable postsynaptic lattice, in allowing proteins to enter this lattice, and in reorganizing this structure in response to plasticity-inducing stimuli.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Domínios PDZ/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/genética , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Imunossupressores/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipoilação/fisiologia , Proteínas de Membrana/genética , Mutação/genética , N-Metilaspartato/farmacologia , Técnicas de Cultura de Órgãos , Domínios PDZ/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Transporte Proteico/fisiologia , Ratos , Receptores de AMPA/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sinapses/efeitos dos fármacos , Tacrolimo/farmacologia , Fatores de Tempo , Transfecção/métodosRESUMO
Long-term potentiation (LTP) is accompanied by dendritic spine growth and changes in the composition of the postsynaptic density (PSD). We find that activity-dependent growth of apical spines of CA1 pyramidal neurons is accompanied by destabilization of the PSD that results in transient loss and rapid replacement of PSD-95 and SHANK2. Signaling through PSD-95 is required for activity-dependent spine growth and trafficking of SHANK2. N-terminal PDZ and C-terminal guanylate kinase domains of PSD-95 are required for both processes, indicating that PSD-95 coordinates multiple signals to regulate morphological plasticity. Activity-dependent trafficking of PSD-95 is triggered by phosphorylation at serine 73, a conserved calcium/calmodulin-dependent protein kinase II (CaMKII) consensus phosphorylation site, which negatively regulates spine growth and potentiation of synaptic currents. We propose that PSD-95 and CaMKII act at multiple steps during plasticity induction to initially trigger and later terminate spine growth by trafficking growth-promoting PSD proteins out of the active spine.