PubMed Health⌕ Search

Biomedical subjects

M Sheng

Publications and source records attributed to M Sheng.

At least 37 records · Page 2Linked to original sources
PubMed2000

alpha-actinin-2 in rat striatum: localization and interaction with NMDA glutamate receptor subunits.

Alpha-actinin (alpha-actinin-2) is a protein which links the NR1 and NR2B subunits of N-methyl-D-aspartate (NMDA) glutamate receptors to the actin cytoskeleton. Because of the importance of NMDA receptors in modulating the function of the striatum, we have examined the localization of alpha-actinin-2 protein and mRNA in striatal neurons, and its biochemical interaction with NMDA receptor subunits present in the rat striatum. Using an alpha-actinin-2-specific antibody, we found intense immunoreactivity in the striatal neuropil and within striatal neurons that also expressed parvalbumin, calretinin and calbindin. Conversely, alpha-actinin-2 immunoreactivity was not detected in neurons expressing choline acetyltransferase and neuronal nitric oxide synthase. Dual-label in situ hybridization revealed that the highest expression of alpha-actinin-2 mRNA is in substance P-containing striatal projection neurons. The alpha-actinin-2 mRNA is also present in enkephalinergic projection neurons and interneurons expressing parvalbumin, choline acetyl transferase and the 67-kDa isoform of glutamic acid decarboxylase, but was not detected in somatostatin-expressing interneurons. Immunoprecipitation of membrane protein extracts showed that alpha-actinin-2 is present in heteromeric complexes of NMDA subunits, but is not associated with AMPA receptors in the striatum. A subunit-specific anti-NR1 antibody co-precipitated major fractions of NR2A and NR2B subunits, but only a minor fraction of striatal alpha-actinin-2. Conversely, alpha-actinin-2 antibody immunoprecipitated only modest fractions of striatal NR1, NR2A and NR2B subunits. These data demonstrate that alpha-actinin-2 is a very abundant striatal protein, but exhibits cellular specificity in its expression, with very high levels in substance-P-containing projection neurons, and very low levels in somatostatin and neuronal nitric oxide synthase interneurons. Despite the high expression of this protein in the striatum, only a minority of NMDA receptors are linked to alpha-actinin-2. This interaction may identify a subset of receptors with distinct anatomical and functional properties.

Actinin↗
PubMed2000

Interaction of the postsynaptic density-95/guanylate kinase domain-associated protein complex with a light chain of myosin-V and dynein.

NMDA receptors interact directly with postsynaptic density-95 (PSD-95), a scaffold protein that organizes a cytoskeletal- signaling complex at the postsynaptic membrane. The molecular mechanism by which the PSD-95-based protein complex is trafficked to the postsynaptic site is unknown but presumably involves specific motor proteins. Here we demonstrate a direct interaction between the PSD-95-associated protein guanylate kinase domain-associated protein (GKAP) and dynein light chain (DLC), a light chain subunit shared by myosin-V (an actin-based motor) and cytoplasmic dynein (a microtubule-based motor). A yeast two-hybrid screen with GKAP isolated DLC2, a novel protein 93% identical to the previously cloned 8 kDa dynein light chain (DLC1). A complex containing PSD-95, GKAP, DLC, and myosin-V can be immunoprecipitated from rat brain extracts. DLC colocalizes with PSD-95 and F-actin in dendritic spines of cultured neurons and is enriched in biochemical purifications of PSD. Immunogold electron microscopy reveals a concentration of DLC in the postsynaptic compartment of asymmetric synapses of brain in which it is associated with the PSD and the spine apparatus. We discuss the possibility that the GKAP/DLC interaction may be involved in trafficking of the PSD-95 complex by motor proteins.

Amino Acid Sequence↗
PubMed2000

Developmentally regulated NMDA receptor-dependent dephosphorylation of cAMP response element-binding protein (CREB) in hippocampal neurons.

Developmental changes in the signaling properties of NMDA receptors have been proposed to underlie the loss of plasticity that accompanies brain maturation. Calcium influx through postsynaptic NMDA receptors can stimulate neuronal gene expression via signaling pathways such as the Ras-MAP kinase (MAPK) pathway and the transcription factor cAMP response element-binding protein (CREB). We analyzed MAPK (Erk1/2) and CREB activation in response to NMDA receptor stimulation during the development of hippocampal neurons in culture. At all stages of development NMDA stimulation induced a rapid phosphorylation of CREB on Ser-133 (phospho-CREB). However, the time course of decline in phospho-CREB changed dramatically with neuronal maturation. At 7 d in vitro (7 DIV) phospho-CREB remained elevated 2 hr after strong NMDA stimulation, whereas at 14 DIV phospho-CREB rose only transiently and fell back to below basal levels within 30 min. Moreover, at 14 DIV, but not at 7 DIV, NMDA receptor stimulation induced a dephosphorylation of CREB that previously had been phosphorylated by KCl depolarization or forskolin, suggesting an NMDA receptor-dependent activation of a CREB phosphatase. There was no developmental change in the time course of phospho-CREB induction that followed KCl depolarization or PKA activation, nor was there a developmental change in the time course of phospho-Erk1/2 induced by NMDA receptor activation. We suggest that, during neuronal maturation, NMDA receptor activation becomes linked specifically to protein phosphatases that act on Ser-133 of CREB. Such a developmentally regulated switch in the mode of NMDA receptor coupling to intracellular signaling pathways may contribute to the changes in neural plasticity observed during brain development.

Animals↗
PubMed2000

An intramolecular interaction between Src homology 3 domain and guanylate kinase-like domain required for channel clustering by postsynaptic density-95/SAP90.

Members of the postsynaptic density-95 (PSD-95)/SAP90 family of membrane-associated guanylate kinase (MAGUK) proteins function as multimodular scaffolds that organize protein-signaling complexes at neuronal synapses. MAGUK proteins contain PDZ, Src homology 3 (SH3), and guanylate kinase (GK)-like domains, all of which can function as sites for specific protein-protein interactions. We report here a direct protein-protein interaction between the SH3 domain and the GK region in the PSD-95 family of MAGUKs. The SH3 domain of the PSD-95 family appears to have an atypical binding specificity, because the classical SH3 binding (-P-X-X-P-) motif is absent from the GK domain. Although SH3-GK binding can occur in either an intramolecular or intermolecular manner, the intramolecular mode is preferred, possibly because of additional tertiary interactions available when the SH3 and GK domains are adjacent in the same polypeptide. Mutations disrupting the intramolecular SH3-GK interaction do not interfere with PSD-95 association with the K(+) channel Kv1.4 or with the GK domain-binding protein GKAP. The same mutations, however, inhibit the clustering of Kv1.4 by PSD-95, suggesting that the intramolecular SH3-GK interaction may modulate the clustering activity of PSD-95.

Animals↗
PubMed2000

Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2.

Membrane-associated guanylate kinases (MAGUKs) contain multiple protein-binding domains that allow them to assemble specific multiprotein complexes in particular regions of the cell. CASK/LIN-2, a MAGUK required for EGF receptor localization and signalling in Caenorhabditis elegans, contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3 and guanylate kinase-like domains. In adult rat brain, CASK is concentrated at neuronal synapses and binds to the cell-surface proteins neurexin and syndecan and the cytoplasmic proteins Mint/LIN-10 and Veli/LIN-7. Here we report that, through its guanylate kinase domain, CASK interacts with Tbr-1, a T-box transcription factor that is involved in forebrain development. CASK enters the nucleus and binds to a specific DNA sequence (the T-element) in a complex with Tbr-1. CASK acts as a coactivator of Tbr-1 to induce transcription of T-element containing genes, including reelin, a gene that is essential for cerebrocortical development. Our findings show that a MAGUK which is usually associated with cell junctions has a transcription regulation function.

Animals↗
PubMed2000

CYRL, a novel cytokine receptor-like protein expressed in testis, lung, and spleen.

The interleukin-3 receptor is composed of a ligand-specific alpha subunit (IL-3Ralpha) and a beta subunit (beta(c) or beta(IL3)). Here we report the cloning of a rat brain cDNA transcript with significant homology to IL-3Ralpha, which we have termed CYRL, for CYtokine Receptor-Like protein. A number of conserved motifs identify CYRL as a member of the alpha family of cytokine receptor subunits, but the extracellular domain was too divergent from the mouse IL-3Ralpha sequence to suggest that CYRL is the rat ortholog of IL-3Ralpha. CYRL mRNA expression by Northern blotting was highest in the testis, intermediate in the lung, and modest in spleen, brain, and heart. Antibodies generated against the extracellular domain of CYRL specifically labeled a broad immunoreactive band of M(r) approximately 50,000 in membrane fractions of testis, lung, and spleen. CYRL appears to be a novel cytokine receptor alpha-subunit of unknown function and with no defined ligands.

Amino Acid Sequence↗
PubMed2000

PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering.

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K(+) channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95-induced clusters of Kv1 K(+) channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K(+) channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvbeta subunits. Consistent with a mechanism of plasma membrane channel-PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3-5 microm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Neuronal inwardly rectifying K(+) channels differentially couple to PDZ proteins of the PSD-95/SAP90 family.

Several signaling proteins clustered at the postsynaptic density specialization in neurons harbor a conserved C-terminal PDZ domain recognition sequence (X-S/T-X-V/I) that mediates binding to members of the PSD-95/SAP90 protein family. This motif is also present in the C termini of some inwardly rectifying K(+) (Kir) channels. Constitutively active Kir2 channels as well as G protein-gated Kir3 channels, which are fundamental for neuronal excitability, were analyzed as candidates for binding to PSD-95/SAP90 family members. Therefore C termini of Kir2.1(+), Kir2.3(+), Kir2.4(-), Kir3.1(-), Kir3.2(+), Kir3.3(+) and Kir3.4(-) subunits (+, motif present; -, motif absent) were used as baits in the yeast two-hybrid assay to screen for in vivo interaction with PDZ domains 1-3 of PSD-95/SAP90. In contrast to Kir2.1 and Kir2.3, all Kir3 fragments failed to bind PSD-95 in this assay, which was supported by the lack of coimmunoprecipitation and colocalization of the entire proteins in mammalian cells. A detailed analysis of interaction domains demonstrated that the C-terminal motif in Kir3 channels is insufficient for binding PDZ domains. Kir2.1 and Kir2.3 subunits on the other hand coprecipitate with PSD-95. When coexpressed in a bicistronic internal ribosome entry site expression vector in HEK-293 cells macroscopic and elementary current analysis revealed that PSD-95 suppressed the activity of Kir2.3 channels by >50%. This inhibitory action of PSD-95, which predominantly affects the single-channel conductance, is likely attributable to a molecular association with additional internal interaction sites in the Kir2.3 protein.

Amino Acid Sequence↗
PubMed2000

Association of dystrophin-related protein 2 (DRP2) with postsynaptic densities in rat brain.

The fundamental function of the membrane-associated cytoskeletal proteins dystrophin and utrophin remains unclear. To gain further insights into the dystrophin family of proteins, we have studied dystrophin-related protein 2 (DRP2), whose expression is largely confined to the vertebrate central nervous system. Both human and rat DRP2 are expressed from two alternative but neighboring transcriptional start sites and have simple transcript structures. Antibodies raised against DRP2 detect a characteristic quartet of bands ( approximately 100-120 kDa) in Western blots of rat brain. The DRP2 protein is associated with brain membrane fractions and highly enriched in the postsynaptic density. Immunohistochemistry shows DRP2 to be widely distributed in a punctate pattern on neuronal dendrites and in neuropil, with particular concentration in regions of the brain involved in cholinergic synaptic transmission. Given the presence of utrophin in the cholinergic neuromuscular junction, and perturbations of cholinergic transmission in dystrophin-deficient nematodes, our findings may suggest a role for DRP2 in the organization of central cholinergic synapses.

Animals↗
PubMed2000

Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization.

Redistribution of postsynaptic AMPA- (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-) subtype glutamate receptors may regulate synaptic strength at glutamatergic synapses, but the mediation of the redistribution is poorly understood. We show that AMPA receptors underwent clathrin-dependent endocytosis, which was accelerated by insulin in a GluR2 subunit-dependent manner. Insulin-stimulated endocytosis rapidly decreased AMPA receptor numbers in the plasma membrane, resulting in long-term depression (LTD) of AMPA receptor-mediated synaptic transmission in hippocampal CA1 neurons. Moreover, insulin-induced LTD and low-frequency stimulation-(LFS-) induced homosynaptic CA1 LTD were found to be mutually occlusive and were both blocked by inhibiting postsynaptic clathrin-mediated endocytosis. Thus, controlling postsynaptic receptor numbers through endocytosis may be an important mechanism underlying synaptic plasticity in the mammalian CNS.

Adaptor Protein Complex alpha Subunits↗
PubMed2000

Development of neuron-neuron synapses.

Our understanding of neuronal synapse development has advanced in recent years. The development of glycinergic synapses appears to depend on gephyrin and glycine receptor activity. Molecular characterization of the structure and development of glutamatergic synapses is in progress, but the underlying mechanisms remain unclear. Activity-dependent mechanisms and specific molecules that regulate the morphological development of dendritic spines have recently been identified.

Animals↗
PubMed2000

Distinct molecular mechanisms and divergent endocytotic pathways of AMPA receptor internalization.

Internalization of postsynaptic AMPA receptors depresses excitatory transmission, but the underlying dynamics and mechanisms of this process are unclear. Using immunofluorescence and surface biotinylation, we characterized and quantified basal and regulated AMPA receptor endocytosis in cultured hippocampal neurons, in response to synaptic activity, AMPA and insulin. AMPA-induced AMPA receptor internalization is mediated in part by secondary activation of voltage-dependent calcium channels, and in part by ligand binding independent of receptor activation. Although both require dynamin, insulin- and AMPA-induced AMPA receptor internalization are differentially dependent on protein phosphatases and sequence determinants within the cytoplasmic tails of GluR1 and GluR2 subunits. AMPA receptors internalized in response to AMPA stimulation enter a recycling endosome system, whereas those internalized in response to insulin diverge into a distinct compartment. Thus, the molecular mechanisms and intracellular sorting of AMPA receptors are diverse, and depend on the internalizing stimulus.

Animals↗
PubMed2000

Ligand-gated ion channel interactions with cytoskeletal and signaling proteins.

In recent years, it has become apparent that ligand-gated ion channels (ionotropic receptors) in the neuronal plasma membrane interact via their cytoplasmic domains with a multitude of intracellular proteins. Different classes of ligand-gated channels associate with distinct sets of intracellular proteins, often through specialized scaffold proteins containing PDZ domains. These specific interactions link the receptor channel to the cortical cytoskeleton and to appropriate signal transduction pathways in the cell. Thus ionotropic receptors are components of extensive protein complexes that are likely involved in the subcellular targeting, cytoskeletal anchoring, and localized clustering of the receptors at specific sites on the neuronal surface. In addition to structural functions, receptor-associated proteins can play important roles as activity modulators or downstream effectors of ligand-gated channels.

Animals↗
PubMed2000

The Shank family of scaffold proteins.

Shank proteins make up a new family of scaffold proteins recently identified through their interaction with a variety of membrane and cytoplasmic proteins. Shank polypeptides contain multiple sites for protein-protein interaction, including ankyrin repeats, an SH3 domain, a PDZ domain, a long proline-rich region, and a SAM domain. Binding partners for most of these domains have been identified: for instance, the PDZ domain of Shank proteins interacts with GKAP (a postsynaptic-density protein) as well as several G-protein-coupled receptors. The specific localization of Shank proteins at postsynaptic sites of brain excitatory synapses suggests a role for this family of proteins in the organization of cytoskeletal/ signaling complexes at specialized cell junctions.

Adaptor Proteins, Signal Transducing↗
PubMed2000

[Cataract extraction with rotated-chipping phacoemulsification in the capsule].

OBJECTIVE: To observe the clinical effects of rotated-chipping phacoemulsification in the capsule. METHODS: The operation of continuous circular capsulectomy, nuclear hydrodissection, cataract extraction with rotated-chipping phacoemulsification in capsule and intraocular lens implantation was performed on 215 eyes. RESULTS: Rotated-chipping technique was successfully completed in 215 eyes. In the operation 2 to 4 circles were rotated with the ultrasonic energy 19.3% for 12 seconds on average. The rate of corneal endothelium loss was 7.5%. CONCLUSION: The technique of rotated-chipping phacoemulsification in capsule is safe and rapid which can be used for cataracts with nuclei of various degrees in sclerosis.

Aged↗
PubMed1999

Characterization of the Shank family of synaptic proteins. Multiple genes, alternative splicing, and differential expression in brain and development.

Shank1, Shank2, and Shank3 constitute a family of proteins that may function as molecular scaffolds in the postsynaptic density (PSD). Shank directly interacts with GKAP and Homer, thus potentially bridging the N-methyl-D-aspartate receptor-PSD-95-GKAP complex and the mGluR-Homer complex in synapses (Naisbitt, S., Kim, E., Tu, J. C. , Xiao, B., Sala, S., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569-582; Tu, J. C., Xiao, B., Naisbitt, S., Yuan, J. P., Petralia, R. S., Brakeman, P., Doan, A., Aakalu, V. K., Lanahan, A. A., Sheng, M., and Worley, P. F. (1999) Neuron 23, 583-592). Shank contains multiple domains for protein-protein interaction including ankyrin repeats, an SH3 domain, a PSD-95/Dlg/ZO-1 domain, a sterile alpha motif domain, and a proline-rich region. By characterizing Shank cDNA clones and RT-PCR products, we found that there are four sites for alternative splicing in Shank1 and another four sites in Shank2, some of which result in deletion of specific domains of the Shank protein. In addition, the expression of the splice variants is differentially regulated in different regions of rat brain during development. Immunoblot analysis of Shank proteins in rat brain using five different Shank antibodies reveals marked heterogeneity in size (120-240 kDa) and differential spatiotemporal expression. Shank1 immunoreactivity is concentrated at excitatory synaptic sites in adult brain, and the punctate staining of Shank1 is seen in developing rat brains as early as postnatal day 7. These results suggest that alternative splicing in the Shank family may be a mechanism that regulates the molecular structure of Shank and the spectrum of Shank-interacting proteins in the PSDs of adult and developing brain.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Characterization of glutamate receptor interacting protein-immunopositive neurons in cerebellum and cerebral cortex of the albino rat.

Glutamate receptor interacting protein (GRIP) binds to the C-terminus of the glutamate receptor 2 (GluR2) subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in vitro and may play an important role in the synaptic organization of these receptors. To determine the distribution of GRIP in vivo, GRIP was localized immunocytochemically in cerebellum and cerebral cortex of adult Sprague-Dawley rats. In the cerebellar cortex, GRIP staining was prominent in perikarya and proximal dendrites of Purkinje cells, whereas Golgi cells were stained more weakly. Double labeling revealed that GRIP and GluR2 were colocalized in Purkinje cells but not in Golgi cells. In the cerebral cortex, GRIP-stained dendrites and somata of nonpyramidal neurons were scattered throughout cortical layers, whereas pyramidal cells were only weakly immunopositive. GRIP was especially prominent in a subset of GluR2-containing cells that also expressed a high level of GluR1. The large majority of strongly GRIP-positive cells in neocortex were immunopositive for gamma-aminobutyric acid (GABA), including the overwhelming majority of calbindin-positive cells in superficial cortical layers, most of the parvalbumin-positive cells, and half of the calretinin-positive interneurons. Staining in the neuropil became more punctate after antigen was unmasked with proteinase K. Electron microscopic localization in the cerebral cortex by postembedding immunogold showed that somatic GRIP was associated with rough endoplasmic reticulum and Golgi apparatus. GRIP was seen over the postsynaptic density of axospinous and axodendritic asymmetric synapses and at high levels in dendrites of GABA-positive neurons. The present data support a role for GRIP in anchoring AMPA receptors and suggest that GRIP trafficking may be especially active in GABAergic neurons.

Animals↗