Subcellular localization of the 52,000 molecular weight major postsynaptic density protein

Kelly, Paul; Montgomery, Paul R.

doi:10.1016/0006-8993(82)91202-1

Cited by 64 publications

(45 citation statements)

References 43 publications

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“…When it was originally described, it was postulated that the mPSDp might be a specific marker for the PSD (18,50 (21) have demonstrated that the mPSDp is crosslinked to PSD proteins by disulfide bonds. Thus, disulfide bonds are probably involved in the association of the kinase with the PSD fraction.…”

Section: Discussionmentioning

confidence: 99%

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

Kennedy¹,

Bennett²,

Erondu³

1983

Proc. Natl. Acad. Sci. U.S.A.

516

261

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By three criteria, two biochemical and one immunochemical, the major postsynaptic density protein (mPSDp) is indistinguishable from the 50-kilodalton (kDa) a subunit of a brain calmodulin-dependent protein kinase. First, the two proteins comigrate on NaDodSO4/polyacrylamide gels. Second, iodinated tryptic peptide maps of the two -are identical. Finally, a monoclonal antibody (6G9) that was raised against the protein kinase binds on immunoblots to a single 50 kDa band in crude brain homogenates and to both the a subunit of the purified kinase and the mPSDp from postsynaptic density fractions. The purified kinase holoenzyme also contains a 60-kDa subunit termed 13. A comparison of the peptide map of .8 with the maps of 60-kDa proteins from the postsynaptic density fraction suggests that ,B is present there but is not the only protein present in this molecular weight range. These results indicate that the calmodulin-dependent protein kinase is a major constituent of the postsynaptic density fraction and thus may be a component of type I postsynaptic densities.Many synaptic junctions in the central nervous system contain a prominent specialized structure called the "postsynaptic density" (PSD) (1-5). When viewed by electron microscopy, it is a fibrous, electron-opaque thickening lying opposite the presynaptic terminal, on the cytoplasmic side of the postsynaptic membrane. The morphology of postsynaptic densities is variable. Some are thick [20-60 nm (5)] and appear to cover the entire postsynaptic surface area, whereas others are thin, often discontinuous, patches (1, 6). The former have been termed type I PSDs, while the latter are called-type II (1). Some investigators have suggested that different types of densities are associated with synapses of particular types. For example, type I PSDs most often occur in synapses that are thought to be excitatory (1,7,8), whereas type II PSDs are seen in synapses thought to be inhibitory (9, 10). This hypothesis suggests.that morphologically distinct PSDs may also contain distinct proteins that serve specialized functions associated with their particular transmitter type.In order to understand the structure and function of PSDs, various research groups have developed subcellular fractionation methods to isolate highly enriched preparations of PSDlike material (11-13). These procedures involve osmotic lysis of a subcellular fraction enriched in synaptosomes, followed by purification of junctional membrane complexes and extraction of membrane components by treatment with detergent. The detergent-insoluble residue, purified by density gradient fractionation, consists of electron-opaque, fibrous, disk-shaped structures, 20-60 nm thick and 200-500 nm in diameter (11-15). These structures are -similar in appearance and staining characteristics to type I PSDs in intact fixed tissue, and they are not produced by detergent treatment of other subcellular organelles such as mitochondria or myelin (11). Thus, this subcellular fraction is considered to be highly enriched in type...

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Section: Discussionmentioning

confidence: 99%

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

Kennedy¹,

Bennett²,

Erondu³

1983

Proc. Natl. Acad. Sci. U.S.A.

516

261

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“…We investigated the proteins contained in a broad band at 180 kDa ( Fig. 1) because they have previously been shown to contain tyrosine phosphate (32), to be phosphorylated by a calmodulin-dependent protein kinase (33), and to remain associated with the PSD fraction after extraction with the harsh detergent sarkosyl (18,34). Three proteins associated with this broad band were resolved from one another.…”

Section: Resultsmentioning

confidence: 99%

The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B.

Moon

Apperson

Kennedy

1994

Proc. Natl. Acad. Sci. U.S.A.

299

181

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The postsynaptic density (PSD) is a specialization of the submembranous cytoskeleton that is visible in the electron microscope on the cytoplasmic face ofthe postsynaptic membrane. A subcellular fraction enriched in ructures with the morphology of PSDs contains signal-transduction molecules thought to regulate receptor llization and function in the central nervous system. We have purified a prominent tyrosine-phosphorylated glycoprotein of apparent molecular mass 180 kDa, termed PSD-gplSO, that is highly enriched in the rat forebrain PSD fraction. The sequences of four tryptic peptides generated from the protein reveal that it is the 2B subunit of the N-methyl-D-aspartate (NMDA) Here we show that a 180-kDa glycoprotein highly enriched in the PSD fraction is subunit 2B of the NMDA receptor (NR2B). We show also that NR2B is the most prominently tyrosine-phosphorylated protein in the PSD fraction.Two distinct types of NMDA receptor subunits were identified recently by molecular cloning (20)(21)(22). Like most ionotropic receptors, NMDA receptors are composed of multiple subunits with homologous sequences (23,24). NMDA receptor subunit 1 (termed NR1) has a molecular mass (106 kDa) close to that of the subunits of the AMPA class ofglutamate receptors. In contrast, the NMDA receptor subunits termed 2A, 2B, 2C, and 2D (NR2A, NR2B, NR2C, and NR2D, respectively) are considerably larger, having molecular masses from 135 kDa to 166 kDa. Their larger sizes result from long C termini that are only distantly related to each other in sequence. Phosphorylation sites for protein kinase C have been localized to the extreme C terminus ofthe NR1 subunit (25), suggesting that the long C-terminal domains of the homologous type 2 subunits also extend into the cytosol where they could mediate additional regulation by phosphorylation (25,26). NR1 is expressed throughout the brain and is essential for formation of a receptor channel; whereas, the NR2 subunits are not essential for channel formation, have more restricted expression patterns, and appear to modulate receptor channel kinetics in different ways (22-24). The findings reported here suggest that NR2B associates tightly with the PSD structure and thus may mediate specific interactions with PSD proteins and other cytosolic proteins perhaps through tyrosine phosphorylated domains.Abbreviations: NMDA, N-methyl-D-aspartate; AMPA, a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid; PSD, postsynaptic density; NR1, NR2B, etc., NMDA receptor subunits 1, 2B, etc.; NOG, n-octyl glucoside. 3954The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…Postsynaptic distribution CAM II kinase-cr is particularly enriched in postsynaptic densities Kelly et al, 1984;Ouimet et al, 1984a), and cell fractionation studies that preferentially isolate the postsynaptic densities of asymmetric synapses (Kelly and Cotman,198 1) have shown that the protein is a major constituent of these densities (Kelly and Montgomery, 1982;Kelly and Vernon, 1985). It is not clear from published electron micrographs whether it is also associated with the other of the two principal forms of forebrain synapse, the symmetric synapses (see Peters et al, 1976).…”

Section: Presynaptic Distributionmentioning

confidence: 99%

Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

et al. 1991

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In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin- dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non- GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate. This suggests that the kinase may be differentially engaged in pre- and postsynaptic functions at certain synapses.

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Subcellular localization of the 52,000 molecular weight major postsynaptic density protein

Cited by 64 publications

References 43 publications

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B.

Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

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