Relative and Absolute Quantification of Postsynaptic Density Proteome Isolated from Rat Forebrain and Cerebellum

Abstract

The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD. Molecular & Cellular Proteomics 5:1158 -1170, 2006.In excitatory synapses of the mammalian brain, the postsynaptic density (PSD) 1 is a specialized membrane-associated structure containing a high concentration of glutamate receptors, cell adhesion molecules, and associated scaffold proteins and signaling enzymes (1-3). Glutamate receptors in the PSD are assembled into large protein complexes by binding to PDZ domain-containing scaffold proteins. In a well characterized example, the cytoplasmic C termini of NR2 subunits of the NMDA-type glutamate receptor interact with the PDZ domains of the PSD-95 family of scaffold proteins, which are highly enriched in the PSD (2). PSD-95 in turn binds to cytoplasmic signaling proteins such as SynGAP, the synaptic GTPase-activating protein (GAP) for Ras/Rap small GTPases (4, 5). Assembly of such protein complexes facilitates specific coupling of postsynaptic receptors to trafficking mechanisms and downstream signaling pathways that control synaptic strength, cytoskeletal rearrangements, and nuclear responses (1). Many if not most of the protein constituents of the PSD are dynamically influenced by synaptic activity via mechanisms such as protein phosphorylation, local translation, ubiquitination, degradation (6, 7), and protein translocation into and out of synapses (8). Altered composition and structural remodeling of the PSD are believed to play critical roles in the formation/elimination and plasticity of synapses. Because it is amenable to biochemical purification and because of its compact size (a few hundred nanometers in diameter and 20 -40 nm thick), the PSD is a highly suitable "o...

Subcellular organization of UBE3A in neurons

“…We found UBE3A in the cytoplasm of dendritic spines at levels equivalent to that found in dendritic shafts. Labeling was rarely directly over PSDs, consistent with findings from biochemical fractionation studies (Gustin et al, 2010) and with the negative findings of several proteomic studies (Cheng et al, 2006;Collins et al, 2006;Bayes et al, 2012), and thus is unlikely to reflect an inaccessibility of PSDs to UBE3A antibodies. Despite a relatively low concentration in PSDs, UBE3A may nevertheless participate locally in postsynaptic regulation, perhaps by regulating the turnover of SAP97 and other synaptic proteins within the cytoplasm of dendritic spines (Matsumoto et al, 2006;Li et al, 2014).…”

An allosteric model of calmodulin explains differential activation of PP2B and CaMKII

“…However, although PP2B has been detected in PSD fractions (reviewed in ref. 33), CaMKII is far more abundant and, in fact, constitutes a major PSD protein (34,35). Together with our allosteric model, this information can explain the differential activation of PP2B at low calcium concentrations and of CaMKII at high calcium concentrations.…”

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

“…4). SynGAP1 is a postsynaptic density protein that exhibits nearly equimolar abundance in forebrain as PSD-95 (68), and loss of SynGAP1 through targeted mutation is deleterious to barrel cortex formation during development (69) and results in learning deficits (59,70). Furthermore, SynGAP1 mRNA has been shown previously to be spatially concentrated in layers II and V of P35 mice (69).…”