Identification of Proteins in the Postsynaptic Density Fraction by Mass Spectrometry

Walikonis, Randall S.; Jensen, Ole Mejlhede; Mann, Matthias; Provance, David William; Mercer, John A.; Kennedy, Mary B.

doi:10.1523/jneurosci.20-11-04069.2000

Cited by 374 publications

(329 citation statements)

References 113 publications

Supporting

Mentioning

307

Contrasting

Unclassified

Order By: Relevance

“…Accordingly, enzymes involved in these pathways are detected in our PSD preparation. It has been demonstrated that CaMKII␣ is the major protein constituent of the PSD fraction (18). In the present study CaMKII␣ is detected as 6 distinct spots that differ slightly in M r and/or pI, suggesting that they may be differentially post-translationally modified.…”

Section: Discussionsupporting

confidence: 52%

See 1 more Smart Citation

Proteomics Analysis of Rat Brain Postsynaptic Density

Hornshaw

Schors

et al. 2004

Journal of Biological Chemistry

235

View full text Add to dashboard Cite

The postsynaptic density contains multiple protein complexes that together relay the presynaptic neurotransmitter input to the activation of the postsynaptic neuron. In the present study we took two independent proteome approaches for the characterization of the protein complement of the postsynaptic density, namely 1) two-dimensional gel electrophoresis separation of proteins in conjunction with mass spectrometry to identify the tryptic peptides of the protein spots and 2) isolation of the trypsin-digested sample that was labeled with isotope-coded affinity tag, followed by liquid chromatography-tandem mass spectrometry for the partial separation and identification of the peptides, respectively. Functional grouping of the identified proteins indicates that the postsynaptic density is a structurally and functionally complex organelle that may be involved in a broad range of synaptic activities. These proteins include the receptors and ion channels for glutamate neurotransmission, proteins for maintenance and modulation of synaptic architecture, sorting and trafficking of membrane proteins, generation of anaerobic energy, scaffolding and signaling, local protein synthesis, and correct protein folding and breakdown of synaptic proteins. Together, these results imply that the postsynaptic density may have the ability to function (semi-) autonomously and may direct various cellular functions in order to integrate synaptic physiology.The majority of excitatory neurotransmission in the brain occurs via glutamatergic synapses. In the presynaptic element of the synapse, specialized secretion machinery determines the activity-dependent membrane fusion of glutamate-containing vesicles and the release of transmitter into the synaptic cleft. In the postsynaptic element, glutamate receptors and downstream signal transduction are organized by the protein assembly of the postsynaptic density (PSD). 1 Both the presynaptic release machinery and the PSD are electron-dense assemblies (1, 2), in which proteins are thought to be organized into distinct functional complexes (3-5) that may be dynamically regulated by neuronal activity (6 -8). The modulation of this molecular architecture of the synapse is at the basis of synaptic plasticity (6 -8), and aberrations thereof may underlie neuronal disorders.In view of the importance of the PSD in glutamatergic neurotransmission and its involvement in neuroplasticity, considerable efforts have been made to identify its protein constituents as a prelude to understand the molecular basis of PSD functioning. In the past several years, yeast two-hybrid technology has been extensively used to characterize proteins that interact with the glutamate receptors and may constitute core elements of the PSD involved in the regulation of receptor trafficking and signaling (reviewed in Refs. 9 -11). Based largely on these studies a protein-protein interaction map of the PSD has emerged. In brief, the model posits that the postsynaptic receptor complexes are localized by scaffolding proteins such as the s...

show abstract

Section: Discussionsupporting

confidence: 52%

“…One of the first applications of proteome research in neuroscience was aimed at the characterization of novel PSD proteins (18). The PSD fraction was isolated using a standard protocol developed by Carlin et al (19).…”

mentioning

confidence: 99%

Proteomics Analysis of Rat Brain Postsynaptic Density

Hornshaw

Schors

et al. 2004

Journal of Biological Chemistry

235

View full text Add to dashboard Cite

show abstract

“…This complex, together with hsp90, has been proposed to control neurotransmitter release (Sakisaka et al, 2002). Complementary, two members of the hsp90 cochaperone machinery, hsp70 and hsp40, have been found at the postsynaptic density (Suzuki et al, 1999;Walikonis et al, 2000;Moon et al, 2001), although the function of this postsynaptic complex was unknown. Here, using electrophysiological techniques, we show that hsp90 plays important roles in synaptic transmission at both the presynaptic and postsynaptic compartments through independent mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Independent Functions of hsp90 in Neurotransmitter Release and in the Continuous Synaptic Cycling of AMPA Receptors

Gerges¹,

Tran²,

Backos³

et al. 2004

J. Neurosci.

View full text Add to dashboard Cite

The delivery of neurotransmitter receptors into synapses is essential for synaptic function and plasticity. In particular, AMPA-type glutamate receptors (AMPA receptors) reach excitatory synapses according to two distinct routes: a regulated pathway, which operates transiently during synaptic plasticity, and a constitutive pathway, which maintains synaptic function under conditions of basal transmission. However, the specific mechanisms that distinguish these two trafficking pathways are essentially unknown. Here, we evaluate the role of the molecular chaperone hsp90 (heat shock protein 90) in excitatory synaptic transmission in the hippocampus. On one hand, we found that hsp90 is necessary for the efficient neurotransmitter release at the presynaptic terminal. In addition, we identified hsp90 as a critical component of the cellular machinery that delivers AMPA receptors into the postsynaptic membrane. Using the hsp90-specific inhibitors radicicol and geldanamycin, we show that hsp90 is required for the constitutive trafficking of AMPA receptors into synapses during their continuous cycling between synaptic and nonsynaptic sites. In contrast, hsp90 function is not required for either the surface delivery of AMPA receptors into the nonsynaptic plasma membrane or for the acute, regulated delivery of AMPA receptors into synapses during plasticity induction (long-term potentiation). The synaptic cycling of AMPA receptors was also blocked by an hsp90-binding tetratricopeptide repeat (TPR) domain, suggesting that the role of hsp90 in AMPA receptor trafficking is mediated by a TPR domaincontaining protein. These results demonstrate new roles for hsp90 in synaptic function by controlling neurotransmitter release and, independently, by mediating the continuous cycling of synaptic AMPA receptors.

show abstract

“…The isolated PSDs appeared to retain the morphological characteristics of their brain counterparts. Isolated PSD samples contain more than 30 proteins (Siekevitz 1985;Walsh and Kurue 1992;Kennedy 1993;Serafini 1997;Walikonis et al 2000). Based on the functions of the proteins found in the PSD, it has been proposed that the PSD plays important roles in (i) the clustering of neurotransmitter receptors in the postsynaptic membrane, (ii) placement of intracellular signaling molecules in close proximity to neurotransmitter receptors, and (iii) the adhesion of presynaptic and postsynaptic components into an integrated synaptic unit (for reviews, see Siekevitz 1985;Ziff 1997;Kennedy 2000).…”

mentioning

confidence: 99%

Structural role of zinc ions bound to postsynaptic densities

Jan

Chen

Tsai

et al. 2002

Journal of Neurochemistry

View full text Add to dashboard Cite

Postsynaptic densities (PSDs) isolated from porcine cerebral cortices are large aggregates consisting of more than 30 different proteins. Inductively coupled plasma-mass spectrometric analyses revealed that isolated PSDs contained zinc at a concentration of 4.1 nmol per mg protein. Treatment with 8 M urea lead to dissociation of the PSDs into small components and, concomitantly, depletion of most of their bound zinc. After removal of the urea by dialysis, urea-dissociated PSD proteins did not reassemble into aggregates by themselves. Adding ZnCl 2 to urea-treated PSD samples resulted in the assembly of urea-dissociated proteins into large aggregates with morphology and protein composition closely resembling those of the original PSDs. Mg 2+ , Ca 2+ , Co 2+ , Cd 2+ , Cu 2+ , Mn 2+ , Fe 3+ , K + and Na + ions at higher concentrations also induced the aggregation of urea-dissociated PSD protein. The structures of the K + -, Na + -, Mg 2+ -and Ca 2+ -induced aggregates were distinct from that of the original PSDs. Our results indicate that the structure of the PSD could be disassembled and reassembled under in vitro conditions. They further suggest that Zn 2+ ions, by binding to certain zincbinding proteins, play an important role in the formation and maintenance of the structure of the PSD.

show abstract

Identification of Proteins in the Postsynaptic Density Fraction by Mass Spectrometry

Cited by 374 publications

References 113 publications

Proteomics Analysis of Rat Brain Postsynaptic Density

Proteomics Analysis of Rat Brain Postsynaptic Density

Independent Functions of hsp90 in Neurotransmitter Release and in the Continuous Synaptic Cycling of AMPA Receptors

Structural role of zinc ions bound to postsynaptic densities

Contact Info

Product

Resources

About