Biomedical subjects

J W Gurd

Publications and source records attributed to J W Gurd.

At least 19 recordsLinked to original sources

PubMed2000

Altered interaction between PSD-95 and the NMDA receptor following transient global ischemia.

The postsynaptic density (PSD) is a cytoskeletal specialization involved in the anchoring of neurotransmitter receptors and in regulating the response of postsynaptic neurons to synaptic stimulation. The postsynaptic protein PSD-95 binds to NMDA receptor subunits NR2A and NR2B and to signaling molecules such as neuronal nitric oxide synthase and p135synGAP. We investigated the effects of transient cerebral ischemia on protein interactions involving PSD-95 and the NMDA receptor in the rat hippocampus. Ischemia followed by reperfusion resulted in a decrease in the solubility of the NMDA receptor and PSD-95 in 1% sodium deoxycholate, the decrease being greater in the vulnerable CA1 hippocampal subfield than in the less sensitive CA3/dentate gyrus regions. Solubilization of the kainic acid receptor GluR6/7 and the PSD-95 binding proteins, neuronal nitric oxide synthase and p135synGAP, also decreased following ischemia. The association between PSD-95 and NR2A and NR2B, as indicated by coimmunoprecipitation, was less in postischemic samples than in sham-operated controls. Ischemia also resulted in a decrease in the size of protein complexes containing PSD-95, but had only a small effect on the size distribution of complexes containing the NMDA receptor. The results indicate that molecular interactions involving PSD-95 and the NMDA receptor are modified by an ischemic challenge.

Animals↗

PubMed2000

Altered association of protein tyrosine kinases with postsynaptic densities after transient cerebral ischemia in the rat brain.

Transient cerebral ischemia results in an increase in the tyrosine phosphorylation of proteins associated with postsynaptic densities (PSDs). The authors investigated the possible mechanisms behind this increase by analyzing isolated PSDs for protein tyrosine kinase activity and for the presence of specific tyrosine kinases. Transient (15 minutes) global ischemia was produced in adult rats by four-vessel occlusion, and PSDs were isolated immediately after ischemia or after 20 minutes or 6 hours of reperfusion. Tyrosine phosphorylation of several PSD proteins, including the N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, was enhanced relative to shams after 20 minutes of reperfusion and underwent a further increase between 20 minutes and 6 hours. The ability of intrinsic PSD tyrosine kinase to phosphorylate PSD proteins, including the NMDA receptor, increased threefold after ischemia. Whereas PSD-associated proline-rich tyrosine kinase 2 (PYK2) and gp145TrkB were elevated immediately after the ischemic event, increases in Src and Fyn were not apparent until 6 hours of reperfusion. The level of PSD-associated pp125FAK decreased after ischemia. The results demonstrate that ischemia results in selective changes in the association of protein tyrosine kinases with the PSD which may account for ischemia-induced increases in the tyrosine phosphorylation of PSD proteins.

Animals↗

PubMed1999

The effect of transient global ischemia on the interaction of Src and Fyn with the N-methyl-D-aspartate receptor and postsynaptic densities: possible involvement of Src homology 2 domains.

Transient ischemia increases tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B in the rat hippocampus. The authors investigated the effects of this increase on the ability of the receptor subunits to bind to the Src homology 2 (SH2) domains of Src and Fyn expressed as glutathione-S-transferase-SH2 fusion proteins. The NR2A and NR2B bound to each of the SH2 domains and binding was increased approximately twofold after ischemia and reperfusion. Binding was prevented by prior incubation of hippocampal homogenates with a protein tyrosine phosphatase or by a competing peptide for the Src SH2 domain. Ischemia induced a marked increase in the tyrosine phosphorylation of several proteins in the postsynaptic density (PSD), including NR2A and NR2B, but had no effect on the amounts of individual NMDA receptor subunits in the PSD. The level of Src and Fyn in PSDs, but not in other subcellular fractions, was increased after ischemia. The ischemia-induced increase in the interaction of NR2A and NR2B with the SH2 domains of Src and Fyn suggests a possible mechanism for the recruitment of signaling proteins to the PSD and may contribute to altered signal transduction in the postischemic hippocampus.

Amino Acid Sequence↗

PubMed1999

Hypoxia-ischemia in perinatal rat brain induces the formation of a low molecular weight isoform of striatal enriched tyrosine phosphatase (STEP).

Protein tyrosine phosphatases play a critical role in controlling tyrosine phosphorylation levels of proteins. Ischemia induces changes in tyrosine phosphorylation. As part of our investigations of the mechanisms responsible for these changes, we studied the effects of cerebral hypoxia-ischemia in 7-day-old (P7) and P21 rat brains on expression of the STEP (striatal enriched phosphatase) family of protein tyrosine phosphatases. P7 and P21 rats were subjected to unilateral hypoxia-ischemia, and brains were analyzed at various intervals of recovery for the presence of STEP. Hypoxia-ischemia induced the formation of a low Mr isoform of STEP, STEP33, in the ipsilateral (damaged) hemisphere but not in the contralateral (undamaged) side. STEP33 produced as a result of ischemia was located exclusively in the cell soluble fraction. In P21 rats, the ischemia-induced elevation in STEP33 was delayed relative to P7 rats. STEP33 was produced by digestion of postsynaptic densities with calpain I and by exposure of NT2/D1 cells expressing STEP to the calcium ionophore A23187. The results suggest that ischemia-induced calcium influx results in the calcium-dependent proteolysis of membrane-associated STEP61 and the concomitant release of STEP33 into the cytoplasm.

Aging↗

PubMed1999

Calcium-dependent cleavage of striatal enriched tyrosine phosphatase (STEP).

Striatal enriched phosphatase (STEP) is a family of protein tyrosine phosphatases enriched within the CNS. A member of this family, STEP61, is a membrane-associated protein located in postsynaptic densities of striatal neurons. In this study, we demonstrate that STEP61, is cleaved into smaller isoforms. To clarify the mechanism of cleavage, STEP61 was transiently expressed in NT2/D1 neuronal precursor cells. Exposure of transfected cells to the calcium ionophore, A23187, or to thapsigargin resulted in the rapid cleavage of STEP61. Pretreatment with the calpain inhibitor, calpeptin, or EGTA prevented proteolysis. One of the cleavage products has a relative molecular mass of 33 kDa (STEP33). A protein with the identical mobility is detected following calpain treatment of STEP61 fusion protein or postsynaptic densities purified from rat striatum. Exposure of primary neuronal cultures to glutamate also led to a significant increase in the concentration of a low molecular weight form of STEP. Taken together, these results suggest that in response to a rapid influx of calcium, STEP61, is proteolytically cleaved by calpain, leading to the release of a smaller isoform. This model may explain the rapid appearance of STEP33 in response to transient hypoxia-ischemia in the brain as cells attempt to counter the increase in tyrosine phosphorylation levels following neuronal insults.

Animals↗

PubMed1998

Identification of lectin-purified neural glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA receptor subunits: conservation of glycosylation at the synapse.

The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (pan-AMPA) receptors. Sensitivities to the glycosidases peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H on both western blots and silver-stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for approximately 50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.

Amidohydrolases↗

PubMed1998

Decreased expression and functionality of NMDA receptor complexes persist in the CA1, but not in the dentate gyrus after transient cerebral ischemia.

The authors investigated the gene expression of the NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptor and the functional electrophysiologic activity of NMDA receptor complexes in the vulnerable CA1 and less vulnerable dentate gyrus subfields of the rat hippocampus at different times after transient cerebral ischemia. Decreased expression for both subtypes was observed in both the CA1 subfield and dentate granule cell layer at early times after challenge; however, the decreased expression in the dentate granule cell layer was reversible because mRNA levels for both the NR2A and NR2B subtypes recovered to, or surpassed, sham-operated mRNA levels by 3 days postchallenge. No recovery of expression for either subtype was observed in the CA1 subfield. The functional activity of NMDA receptor complexes, as assessed by slow field excitatory postsynaptic potentiations (slow f-EPSP) in CA1 pyramidal neurons, was maintained at 6 hours postchallenge; however, this activity was diminished greatly by 24 hours postchallenge, and absent at 7 days postchallenge. A similar pattern was observed for the non-NMDA receptor-mediated fast f-EPSP. In dentate granule neurons, however, no significant change in NMDA receptor-mediated slow f-EPSP from sham control was observed at any time after insult. The non-NMDA receptor-generated fast f-EPSPs also were maintained at all times postinsult in the dentate gyrus. These results illustrate that the activity of NMDA receptors remains functional in dentate granule neurons, but not in the pyramidal neurons of the CA1 subfield, at early and intermediate times after transient cerebral ischemia, and suggest that there is a differential effect of ischemia on the glutamatergic transmission systems in these two hippocampal subfields.

Animals↗

PubMed1997

Protein tyrosine phosphorylation: implications for synaptic function.

The phosphorylation of proteins on tyrosine residues, initially believed to be primarily involved in cell growth and differentiation, is now recognized as having a critical role in regulating the function of mature cells. The brain exhibits one of the highest levels of tyrosine kinase activity in the adult animal and the synaptic region is particularly rich in tyrosine kinases and tyrosine phosphorylated proteins. Recent studies have described the effects of tyrosine phosphorylation on the activities of a number of proteins which are potentially involved in the regulation of synaptic function. Furthermore, it is becoming apparent that tyrosine phosphorylation is involved in the modification of synaptic activity, such as occurs during depolarization, the induction of long-term potentiation or long-term depression, and ischemia. Changes in the activities of tyrosine kinases and/or protein tyrosine phosphatases which are associated with synaptic structures may result in altered tyrosine phosphorylation of proteins located at the synapse leading to both short-term and long-lasting changes in synaptic and neuronal function.

Animals↗

PubMed1997

The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

Adenosine Triphosphate↗

PubMed1997

Transient ischemia differentially increases tyrosine phosphorylation of NMDA receptor subunits 2A and 2B.

Activation of the N-methyl-D-aspartate (NMDA) receptor has been implicated in the events leading to ischemia-induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four-vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti-tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit-specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23-29-fold relative to sham-operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia-induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.

Animals↗

PubMed1997

Transient global ischemia alters NMDA receptor expression in rat hippocampus: correlation with decreased immunoreactive protein levels of the NR2A/2B subunits, and an altered NMDA receptor functionality.

We investigated the gene expression levels, the immunoreactive protein prevalence, and the functional activity of N-methyl-D-aspartate (NMDA) receptor complexes at early times after severe global ischemia challenge in rats. The mRNA expression levels for the NR2A and NR2B subunits of NMDA receptors changed to different degrees within different subregions of the hippocampus after reperfusion with respect to sham-operated control. No significant change in expression was observed in the vulnerable CA1 subfield at or before 6 h after challenge for either receptor subunit, although changes in expression in other hippocampal subfields were observed. At 12 and 24 h after challenge, significant decreases in expression for both subunits were found in the vulnerable CA1 subfield, as well as in other hippocampal regions. At the protein level, a significant decrease in the amount of NR2A/NR2B immunoreactivity in the total hippocampus was observed at both 6 and 24 h after reperfusion compared with sham control. Electrophysiological assessment of single-channel NMDA receptor activity in the CA1 subfield indicates that the main conductance state of NMDA receptor channels is maintained 6 h after challenge, although by 18-24 h after challenge, this main conductance state is rarely observed. The NMDA receptor component of the excitatory postsynaptic field potential was found to be significantly diminished from sham control 24 h after challenge, such that only approximately 10% of the sham response remained, but was not significantly altered from sham control at 6 h after challenge. These results indicate that decreases in the expression levels, the immunoreactive protein prevalence, and that alterations in the functionality of NMDA receptors occur in the hippocampus at early times after severe transient global ischemia.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1996

Enhanced tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate receptor in long-term potentiation.

Both serine/threonine and tyrosine phosphorylation of receptor proteins have been implicated in the process of long-term potentiation (LTP), but there has been no direct demonstration of a change in receptor phosphorylation after LTP induction. We show that, after induction of LTP in the dentate gyrus of anesthetized adult rats, there is an increase in the tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate (NMDA) receptor (NR2B), as well as several other unidentified proteins. Tyrosine phosphorylation of NR2B was measured in two ways: binding of antiphosphotyrosine antibodies (PY20) to glycoprotein(s) of 180 kDa (GP180) purified on Con A-Sepharose and binding of anti-NR2B antibodies to tyrosine-phosphorylated proteins purified on PY20-agarose. Three hours after LTP induction, anti-NR2B binding to tyrosine phosphorylated proteins, expressed as a ratio of tetanized to control dentate (Tet/Con), was 2.21 +/- 0.50 and PY20 binding to GP180 was 1.68 +/- 0.16. This increase in the number of tyrosine phosphorylated NR2B subunits occurred without a change in the total number of NR2B subunits. When the induction of LTP was blocked by pretreatment of the animal with the NMDA receptor antagonist MK801, the increase in PY20 binding to GP180 was also blocked (Tet/Con = 1.09 +/- 0.26). The increased PY20 binding to GP180 was also apparent 15 min after LTP induction (Tet/Con = 1.41 +/- 0.16) but not detectable 5 min after LTP induction (Tet/Con = 1.01 +/- 0.19). These results suggest that tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP.

Amino Acid Sequence↗

PubMed1996

SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain.

Although extracellular matrix (ECM) components have been shown to play important roles in the development of the CNS, expression generally decreases in the adult brain. This study examines the expression of the SPARC-related glycoprotein SC1 in the rat brain during postnatal development and in the adult. In situ hybridization analysis indicates that expression of SC1 mRNA increases in a caudal to rostral manner as postnatal neural development proceeds and is found at near maximal levels in the adult brain. SC1 mRNA is expressed in glial-enriched areas of the brain at postnatal day 1 (P1) and P5. Between P10 and P20, SC1 mRNA increases in neuron-enriched regions of the hippocampus, dentate gyrus, and cerebral cortex. Immunohistochemistry in the adult shows that SC1 protein is localized to neurons in these regions and to scattered glial cells. Subcellular fractionation demonstrates that the SC1 116/120 kDa doublet is associated with synaptosomes. SC1 is present in the aqueous phase following extraction of membranes with TX-114, suggesting that it is not a transmembrane protein, a property consistent with other adult brain ECM components. Furthermore in cerebellar granule cells grown in culture, high levels of the 120 kDa component are secreted into the media. These results are consistent with the hypothesis that SC1 is an ECM glycoprotein expressed in both the developing and adult brain.

Animals↗

PubMed1995

Tyrosine phosphorylation in a model of ischemia using the rat hippocampal slice: specific, long-term decrease in the tyrosine phosphorylation of the postsynaptic glycoprotein PSD-GP180.

The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M(r) 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M(r) 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro "ischemia" was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N2 instead of O2 and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca2+ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca2+ was omitted from the incubation media.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Tyrosine phosphorylation of glycoproteins in the adult and developing rat brain.

The tyrosine phosphorylation of glycoproteins in the adult and developing rat brain was investigated. Immunoblotting with anti-tyr(P) antibodies identified a glycoprotein with an apparent Mr of 180,000 (GP180) as the major tyrosine-phosphorylated protein in the concanavalin A (con A)-binding fraction prepared from forebrain homogenates. This glycoprotein had the same electrophoretic mobility as the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. Tyrosine-phosphorylated GP180 was enriched 24-fold in isolated PSDs relative to homogenates. Digestion with endoglycosidase F/N-glycosidase F demonstrated that GP180 present in total homogenates and PSD-GP180 present in isolated PSDs contained similar amounts of N-linked oligosaccharide suggesting that they are the same glycoprotein. The tyrosine phosphorylation of GP180 in homogenates varied between brain regions with the highest levels occurring in cortical areas and the amygdala and low or undetectable amounts being present in hindbrain regions. Incubation of homogenates with adenosine triphosphate (ATP) resulted in the tyrosine phosphorylation of GP180 in all regions examined except the cerebellum and identified a second con A-binding glycoprotein, GP110, which was phosphorylated on tyrosine. GP180 was not phosphorylated on tyrosine following the incubation of cerebellar homogenate, synaptic membranes, or PSDs and ATP. Tyr(P)-GP180 was not detected prior to the onset of synaptogenesis, increased in parallel with the formation of synapses during the first 4 weeks of postnatal development of the frontal cortex and hippocampus, and then decreased 50-60% to adult levels. The results suggest that GP180 corresponds to the PSD glycoprotein PSD-GP180 and are consistent with a role for this glycoprotein in synaptic development and signal transduction at the synapse.

Animals↗

PubMed1994

Developmental expression in the rat cerebellum of SC1, a putative brain extracellular matrix glycoprotein related to SPARC.

In the nervous system, extracellular matrix (ECM) molecules have been shown to have effects on cell migration, process outgrowth and the survival of neurons. Recently we have described the molecular cloning of SC1, a putative brain extracellular matrix glycoprotein, showing partial similarity to the ECM glycoprotein SPARC/osteonectin. We have now examined the expression of SC1 during the development of the rat cerebellum at both the protein and mRNA levels. Our results indicate that SC1 is both temporally and spatially regulated during this process. Bergmann glial cells express SC1 mRNA and the resultant protein is deposited along the length of their radial fibres during the process of granule cell migration in the developing cerebellum. SC1 mRNA and protein is also found in the adult cerebellum, concentrated in the Bergmann glial cells and their radial processes, indicating that this putative ECM molecule continues to play roles in the central nervous system after migration and proliferative events have ceased.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed1994

Expression of PAC 1, an epitope associated with two synapse-enriched glycoproteins and a neuronal cytoskeleton-associated polypeptide in developing forebrain neurons.

The monoclonal antibody PAC 1 (postsynaptic density and cytoskeleton enriched) recognizes an epitope present on two postsynaptic density-enriched glycoproteins of 130,000 (postsynaptic density-enriched glycoprotein 130) and 117,000 mol. wt (postsynaptic density-enriched glycoprotein 117), and a cytoskeleton-enriched polypeptide of 155,000 mol. wt (cp155). The PAC 1 antibody has been used to study the development of the PAC 1 antigens in the developing rat forebrain in vivo and in tissue culture. cp155 is detected by embryonic day 14 and its level continues to rise until the sixth postnatal week. Postsynaptic density-enriched glycoproteins 130 and 117 are also expressed in embryonic brain although the level of postsynaptic density-enriched glycoprotein 130 initially increases more rapidly than that of postsynaptic density-enriched glycoprotein 117. Peak values are observed at postnatal days 4 (postsynaptic density-enriched glycoprotein 117) and 9 (postsynaptic density-enriched glycoprotein 130). The level of post synaptic density-enriched glycoprotein 117 subsequently decreases to some 50% of the peak value by postnatal day 42. Immunocytochemical studies show that PAC 1 immunoreactivity in developing cerebral cortex, detectable by postnatal day 0, is primarily associated with the perikarya and dendrites of pyramidal cells. The immunoreactivity develops as patches of PAC 1-positive neurons, uniform staining of the cortex only being fully established after postnatal day 9. Double-immunofluorescence labelling studies of forebrain cultures prepared from embryonic day 18 animals shows that many, but not all, growth-associated protein 43-positive neurons exhibit PAC 1 immunoreactivity. Some non-neuronal cells also stain with the PAC 1 monoclonal antibody. The growth cones of cultured neurons exhibit PAC 1 immunoreactivity and the PAC 1 antigens are detected on immunodeveloped western blots of isolated growth cones. The PAC 1 epitope is intracellular, but immunoreactivity does not co-localize with F-actin as detected by rhod-amine-phalloidin or with tubulin immunoreactivity. Postsynaptic density-enriched glycoprotein 130 is readily detected on PAC 1 immunodeveloped western blots of forebrain cultures maintained for up to 14 days in vitro. Postsynaptic density-enriched glycoprotein 117 is only poorly expressed by these cultures. The PAC 1 glycoproteins are present in forebrain synaptic membranes and postsynaptic densities at an early stage of development. The synaptic membrane level of postsynaptic density-enriched glycoprotein 130 and postsynaptic density-enriched glycoprotein 117 increases markedly between postnatal days 3 and 8. The level of both glycoproteins detected in postsynaptic densities remain virtually constant from postnatal days 9-90. These results are consistent with functional roles for these molecules in neuronal and synapse development.

Animals↗

PubMed1992

Molecular cloning of a novel mRNA using an antibody directed against synaptic glycoproteins.

It has been suggested by a number of investigators that glycoproteins may play a role in the development and/or maintenance of synapses in the mammalian CNS. For many synaptic glycoproteins, however, little precise structural or functional information is available. In an effort to isolate probes specific to individual glycoproteins, we have screened a rat brain cDNA expression library with a mixed polyclonal antibody directed against concanavalin A-binding synaptic junctional glycoproteins. Using this approach, we have previously reported the cloning of SC1, a putative extracellular matrix glycoprotein found in adult brain (Johnston et al., Neuron 2:165-176, 1990). We now report the cloning and characterization of a second novel cDNA, which has been designated SC2. Northern blots show that this cDNA recognizes a 1.2-kb mRNA that is present throughout postnatal development in the rat. It is expressed at high levels in brain and is also found at lower levels in several other tissues. In situ hybridization suggests that the SC2 mRNA is strongly expressed by many types of neurons. Sequence data reveals a single open reading frame in the cDNA, encoding a putative hydrophobic protein with a calculated molecular weight of 36.1 kDa. Sequence analysis reveals some similarity between SC2 and 5 alpha-reductase, a microsomal membrane protein important in testosterone metabolism.

Amino Acid Sequence↗