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PubMed1997

Rapsyn and agrin slow the metabolic degradation of the acetylcholine receptor.

Rapsyn is a 43-kDa cytoplasmic protein that clusters nicotinic acetylcholine receptors (AChR) in the postsynaptic membrane. Here we examine the effect of rapsynmediated AChR clustering on the metabolic stability of the AChR. When transfected into QT-6 fibroblasts, cell surface AChRs (alpha, beta, epsilon, and delta subunit combination) pulse labeled with 125I-alpha-bungarotoxin were degraded with a half-life of 16.4 +/- 1.1 h (mean +/- SEM). Cotransfection of rapsyn with AChR caused extensive AChR clustering and increased AChR half-life to 20.5 +/- 1.0 h. Anti-AChR antibodies such as mab 35 cause an increased AChR degradation often associated with myasthenia gravis: 80.8 +/- 2.5% of AChRs labeled at zero time were degraded over a 12-h period. Contransfection of rapsyn reduced this AChR loss to 66.4 +/- 3.8%. Rapsyn also reduced normal AChR degradation, from 53.2 +/- 2.1 to 44.2 +/- 2.2%. Muscle cell lines from wild-type myotubes displayed few AChR clusters, but treatment with neural agrin increased the number of AChR clusters 30-fold. Clustering was accompanied by reductions in AChR degradation (both in the presence and absence of mab 35) similar in magnitude to those produced by overexpression of rapsyn in QT-6 cells. In rapsyn-deficient myotubes, treatment with neural agrin neither caused AChR clustering nor reduced AChR degradation. Thus neural agrin may slow AChR degradation by inducing the rapsyn-dependent clustering of AChRs.

Agrin↗
PubMed1997

A role of midkine in the development of the neuromuscular junction.

Midkine (MK) is a member of a family of developmentally regulated neurotrophic and heparin-binding growth factors. It is expressed during the midgestation period in a retinoid-acid dependent manner during embryogenesis in the mouse. In vitro, it promotes neurite outgrowth from spinal cord neurons and cell migration. It expression is strongest in the central nervous system, thus suggesting a function for this protein in neural development. In this study, the role of MK in synaptogenesis was examined in the Xenopus system. A Xenopus MK cDNA was cloned from an embryonic library encompassing neurulation and synaptogenesis stages. By Northern blot analysis, MK mRNA was detected from the onset of neurulation and throughout the stages of synaptogenesis in the Xenopus embryo. This suggests that MK is also an important growth regulator in Xenopus embryogenesis. To study the function of MK in the development of the neuromuscular junction (NMJ), fusion proteins were made and their ability to induce the formation of acetylcholine receptor (AChR) clusters in cultured muscle cells was studied. Beads coated with MK strongly induce AChR clustering. When nerve-muscle cocultures were labeled with antibodies made against the MK fusion protein, MK immunoreactivity was detected at the NMJ. Unlike heparin-binding growth-associated molecule (HB-GAM), another member of this growth factor family, MK expression cannot be detected in the muscle but is present in spinal cord neurites. Consistent with these in vitro data is the observation that MK mRNA is only localized in the central nervous system but the protein is deposited at the intersomitic junction where the NMJ is located in vivo. Exogenously applied MK does bind to the heparan sulfate proteoglycan on the surface of Xenopus muscle cells. Agrin, a heparan-sulfate proteoglycan that induces the formation of AChR clusters in cultured muscle cells, binds strongly to MK. Bath application of MK in conjunction with agrin results in a change in the pattern of AChR clustering induced by agrin alone. These data suggest that MK is a neuron-derived factor that participates in the signal transduction process during NMJ development.

Agrin↗
PubMed2000

Nitric oxide synthase (NOS-1) coclustered with agrin-induced AChR-specializations on cultured skeletal myotubes.

Previously we reported that neuronal nitric oxide synthase type-1 (NOS-1) is expressed in skeletal myotubes in vitro. In the present paper we sought to determine whether agrin-induced membrane specializations known to include the nicotinic acetylcholine receptor (AChR) on cultured myotubes may also contain NOS-1 and related molecules. After treatment with various agrin constructs containing the full C-terminally AChR-clustering domain (fragments N2, N4), but not with fragment C2 (truncated), NOS-1 expressed in the cytosol of mouse C2C12 skeletal myotubes coclustered with AChR, 43K rapsyn, MuSK, and the dystrophin/utrophin glycoprotein-complex (DUGC). Agrin-induced specializations also included coaggregates of N-methyl-d-aspartic acid (NMDA)-receptor, alpha-sodium (NaCh), or Shaker-type K+ channel (KCh)/PSD-95 complexes, and NOS-1. We conclude that agrin is crucial for recruitment of preassembled multimolecular membrane clusters, including AChR, NMDAR, and ion channels linked to NOS-1. Coassembly of NOS-1 to postsynaptic molecules may reflect site-specific NO-signaling pathways in neuromuscular junction formation and functions.

Agrin↗
PubMed2001

A novel mechanism for modulating synaptic gene expression: differential localization of alpha-dystrobrevin transcripts in skeletal muscle.

Alpha-dystrobrevin is a dystrophin-related and -associated protein that is involved in synapse maturation and is required for normal muscle function. There are three protein isoforms in skeletal muscle, alpha-dystrobrevin-1, -2, and -3 that are encoded by the single alpha-dystrobrevin gene. To understand the role of these proteins in muscle we have investigated the localisation and transcript distribution of the different alpha-dystrobrevin isoforms. Alpha-dystrobrevin-1 and -2 are concentrated at the neuromuscular junction and are both recruited into agrin-induced acetylcholine receptor clusters in cultured myotubes. We also demonstrate that all alpha-dystrobrevin mRNAs are transcribed from a single promoter in skeletal muscle. However, only transcripts encoding alpha-dystrobrevin-1 are preferentially accumulated at postsynaptic sites. These data suggest that the synaptic accumulation of alpha-dystrobrevin-1 mRNA occurs posttranscriptionally, identifying a novel mechanism for synaptic gene expression. Taken together, these results indicate that different isoforms possess distinct roles in synapse formation and possibly in the pathogenesis of muscular dystrophy.

Agrin↗
PubMed2001

An alternative amino-terminus expressed in the central nervous system converts agrin to a type II transmembrane protein.

Agrin is a basal lamina-associated heparansulfate proteoglycan that is a key molecule in the formation of the vertebrate neuromuscular junction. The carboxy-terminal part of agrin is involved in its synaptogenic activity. The amino-terminal end of chick agrin consists of a signal sequence, required for the targeting of the protein to the secretory pathway, and the amino-terminal agrin (NtA) domain that binds to basal lamina-associated laminins. The cDNA encoding rat agrin lacks this NtA domain and instead codes for a shorter amino-terminal end. While the NtA domain is conserved in several species, including human, sequences homologous to the amino-terminus of rat agrin have not been described. In this work, we have characterized these amino-terminal sequences in mouse and chick. We show that they all serve as a noncleaved signal anchor that immobilizes the protein in a N(cyto)/C(exo) orientation in the plasma membrane. Like the secreted form, this transmembrane form of agrin is highly glycosylated indicative of a heparansulfate proteoglycan. The structure of the 5' end of the mouse agrin gene suggests that a distinct promoter drives expression of the transmembrane form. Agrin transcripts encoding this form are enriched in the embryonic brain, particularly in neurons. To our knowledge, this is the first example of a molecule that is synthesized both as a basal lamina and a plasma membrane protein.

Agrin↗
PubMed1998

Structural interactions between chemokine receptors, gp120 Env and CD4.

Seven transmembrane segment (7TMS) receptors for chemokines and related molecules have been demonstrated to be essential, in addition to CD4, for HIV and SIV infection. The beta-chemokine receptor CCR5 is the primary, perhaps sole, co-receptor for HIV-1 during the early and chronic phases of infection and supports infection by most primary HIV-1 and many SIV isolates. Late-stage primary and laboratory-adapted HIV-1, HIV-2, and SIV isolates can use other 7TMS receptors. CXCR4 appears especially important in late-stage HIV infection; several related receptors can also be used. The specificity of SIV viruses is similar. Commonalities among these receptors, combined with analyses of mutated molecules, indicate that discrete, conformationally-dependent sites on the chemokine receptors determine their association with the third variable and conserved regions of viral envelope glycoproteins. These studies are useful for elucidating the mechanism and molecular determinants of HIV-1 entry, and of inhibitors to that entry.

Amino Acid Sequence↗
PubMed1999

Tyrosines 60, 64, and 101 of Epstein-Barr virus LMP2A are not essential for blocking B cell signal transduction.

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed on the membrane of B-lymphocytes and blocks B cell receptor (BCR) signaling in EBV-transformed B-lymphocytes in vitro. The LMP2A amino-terminal domain, which is essential for the LMP2A-mediated block of B cell signal transduction, contains eight tyrosine residues. Three of these tyrosine residues (Y74, Y85, and Y112) have been demonstrated to be essential for the LMP2A-mediated block on protein tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression after BCR activation. To investigate the importance of tyrosines at positions 60, 64, and 101 on B cell signaling, EBV recombinants were constructed containing a tyrosine-to-phenylalanine point mutation at amino acid 60, 64, or 101 within LMP2A. Tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression were not affected by any of the tyrosine point mutations after BCR activation. In addition, constitutive phosphorylation of LMP2A was unaffected by any of the tyrosine point mutations. These data indicate that tyrosines 60, 64, and 101 are not essential for the LMP2A-mediated block of B cell signal transduction in transformed cell lines.

Amino Acid Sequence↗
PubMed1987

Mechanisms of receptor-mediated transmembrane signalling.

Although this paper has dealt with general mechanisms whereby a hormonal signal is transmitted across the cell membrane, advances in work with a number of receptors should permit a precision of description of these mechanisms that would have delighted both Langley and Ehrlich. For instance, the detailed sequences now known for the separate subunits of the nicotinic cholinergic receptor and the cellular manipulations made possible by the cloning of the separate subunit genes will make it possible to determine the precise receptor sequence involved either in acetylcholine binding or in ion channel function. The complete sequences and biochemical properties now known for the insulin and EGF-URO receptors to be dealt with in part by a subsequent article (van Obberghen and Gammeltoft, this series) should lay the groundwork for elucidating the transmembrane signalling mechanisms used by the kinase family of growth factor receptors. Continuing work on the structure of the beta-adrenergic receptor, and on the interaction of such receptors with guanine nucleotide regulatory complexes and on the detailed properties of the family of so-called G-proteins and their associated regulatory subunits should unravel the details for a variety of transmembrane signalling reactions. Thus, at least for three basic transmembrane signalling mechanisms: ligand modulated ion transport; ligand-modulated receptor enzyme activity (e.g. tyrosine kinase); and ligand-modulated liberation of cryptic mediators (like the alpha- and beta-subunits of the guanine nucleotide regulatory complexes) one can look forward with excitement to the elucidation in the not-too-distant future of a number of specific biochemical reaction pathways that lead to cell activation.

Animals↗
PubMed1987

Effects of colchicine and cytochalasin B on distribution of concanavalin A receptors in isolated and cultured guinea pig epidermal cells.

Regulation of the distribution of concanavalin A (Con A)/receptor complexes by the cytoskeletal contracture system was studied in guinea pig epidermal cells in suspension and culture using the fluorescence double staining method. After treatment with 100 micrograms/ml of Con A at 37 degrees C for 30 min lectin/receptor complexes were endocytosed by the less-differentiated cells in suspension and by the adherent cells in 1- and 3-day cultures that represent a growing cell fraction. The same treatment resulted in diffuse surface distribution of the complexes in the well-differentiated cells in suspension. Colchicine (10(-5) and 10(-6) M) inhibited internalization of the complexes with resultant diffuse distribution in 60% of the adherent cells in culture. Cytochalasin B (5 and 10 micrograms/ml) not only inhibited endocytosis but promoted formation of surface patchy clumps of the complexes in suspended, less-differentiated cells and cultured adherent cells. The distribution profile was not influenced by these drug treatments in the well-differentiated cells. SDS polyacrylamide gel electrophoresis and autoradiography of 125I-labelled epidermal membranes revealed several Con A-reactive polypeptides common to the cells at various differentiation steps. The progressive decrease in endocytosis and mobility of Con A/receptor complexes was suggested to occur with differentiation. In the germinative cells the distribution of lectin/receptor complexes seemed to be regulated by microfilaments and microtubules.

Animals↗
PubMed1988

Changes in the patching and capping of insulin receptors under the influence of hormonal imprinting.

Binding of fluorescein-isothiocyanate-(FITC)-labeled insulin was followed up in the function of time in Chang liver cells pretreated and not pretreated with insulin. The not pretreated cells showed patching, but no capping of the receptors during the period of study (60 min), whereas the insulin-pretreated cells showed indications of capping already after 10 min. Patching of the insulin receptors was particularly conspicuous at the sites of cell-cell contact (at the intercellular junctions). Supra-nuclear patching occurred earlier in the control cultures, and on it followed the fluorescence of the nuclear chromatin.

Animals↗
PubMed1989

Effect of fluoride on movement of concanavalin A-acceptor molecules of human neutrophils.

The effects of fluoride (F) on neutrophil protuberance formation and induced Con A acceptor molecule migration were assessed microscopically. Below 5 mM, F had little effect on acceptor migration, while it markedly inhibited formation of colchicine-induced protuberances. The anion also increased the rate at which preformed protuberances regressed. Since protuberance formation is enhanced by disassembly of microtubules, these data suggest that F promotes and/or stabilizes microtubule assembly. Microtubule assembly is favored by binding of GTP to tubulin subunits, while GDP binding favors disassembly of microtubules. Since F binds with GDP, forming a new complex that mimics GTP, the anion would be expected to enhance microtubule assembly. Over the same F concentration range, the anion failed to inhibit acceptor polarization, but did inhibit cytochalasin B-enhanced dispersion of prepolarized Con A acceptors, implying that, at low concentrations, F also affected microfilament cycling. Concentrations of F in excess of 5 mM inhibited acceptor migration as well as protuberance formation. At 20 mM, the anion abolished both events, yet at this same concentration F induced neutrophil superoxide generation and degranulation, suggesting that acceptor migration is not a prerequisite for these two neutrophil effector activities.

Actin Cytoskeleton↗
PubMed1990

Oxygen metabolites induced by phorbol myristate acetate increase lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in human polymorphonuclear leukocytes.

To assess the general effects of protein kinase C (PKC) activation on cell membrane receptor mobility in human neutrophilic polymorphonuclear leukocytes (PMNLs), the lateral diffusion of fluoresceinated succinylated wheat germ agglutinin (S-WGA-FITC)-labeled membrane glycoconjugates was measured using fluorescence recovery after photobleaching (FRAP). Activation of PKC was achieved by incubating the PMNLs with different concentrations (5-100 nM) of phorbol myristate acetate (PMA). The membrane effects of dimethyl sulfoxide (DMSO), another possible membrane perturbant, were also studied. We found that PMA treatment (greater than or equal to 10 nM) increased the glycoconjugate diffusion coefficient (D) 2-2.5-fold. The mobile fraction (R) remained constant, around 30%. With DMSO, no effect on the diffusion was seen. The increase in lateral mobility due to cell stimulation with PMA was totally inhibited by catalase (200 units/ml) but only partly with superoxide dismutase (2000 units/ml). Exogenous hydrogen peroxide (0.01-5 mM) had no effect on glycoconjugate mobility in unstimulated cells. We therefore propose that activation of PKC mediates augmented mobility of glycoconjugate receptors in PMNL, a reaction that seems to be critically dependent on formation of reactive oxygen metabolites. The results indicate that endogenous formation of reactive metabolites upon receptor stimulation may have a general effect on receptor mobility.

Catalase↗
PubMed1985

Formation of the active zone at developing neuromuscular junctions in larval and adult bullfrogs.

Development of the presynaptic active zone was studied at neuromuscular junctions with freeze-fracture electron microscopy in larval and adult bullfrogs. In rudimentary larval neuromuscular junctions, clusters of active zone particles were scattered over the P-face of the presynaptic membrane. Vesicle openings were observed at these terminals even though active zone particles lacked the mature pattern of two double rows. Gradually, active zone particles became organized into rows, but they were still randomly located and oriented. Once junctional folds were observed in replicas, developing active zones were located opposite to the folds, as in mature terminals. Multiple terminals occupying the same junctional gutters were also observed. At the end of metamorphosis, most active zones were still immature in appearance and had only grown to one third of their mature length. After metamorphosis, the number of active zone segments aligned at the same junctional fold increased. These discontinuous short active zones then elongated, joined together, and finally formed the mature active zones. Signs suggesting synapse elimination such as disorganization of active zones, absence of intramembrane particles in varicosities, and exposed muscle membranes with patches of acetylcholine receptor aggregates were observed. In some multiply innervated junctions, one terminal had mature active zones with vesicle openings, the other in the same gutter displayed disorganized active zones without vesicle openings, although both terminals showed similar sizes and distributions of background particles. This study suggests that developing active zones, as is the case for regenerating active zones in the adult, are functional before the mature organization is formed. The sequence of development of active zones is also similar to that of regeneration except for the random location and orientation of early active zones in tadpoles. The comparison between regeneration and development further indicates that the process of active zone formation is related to junctional folds and/or associated structures. It is also suggested that synapse elimination may involve degenerative changes in presynaptic membranes, although direct evidence remains to be provided.

Age Factors↗
PubMed1986

Mechanisms of receptor-mediated transmembrane signalling.

Although this paper has dealt with general mechanisms whereby a hormonal signal is transmitted across the cell membrane, advances in work with a number of receptors should permit a precision of description of these mechanisms that would have delighted both Langley and Ehrlich. For instance, the detailed sequences now known for the separate subunits of the nicotinic cholinergic receptor and the cellular manipulations made possible by the cloning of the separate subunit genes will make it possible to determine the precise receptor sequence involved either in acetylcholine binding or in ion channel function. The complete sequences and biochemical properties now known for the insulin and EGF-URO receptors to be dealt with in part by a subsequent article (van Obberghen and Gammeltoft, this series) should lay the groundwork for elucidating the transmembrane signalling mechanisms used by the kinase family of growth factor receptors. Continuing work on the structure of the beta-adrenergic receptor, and on the interaction of such receptors with guanine nucleotide regulatory complexes and on the detailed properties of the family of so-called G-proteins and their associated regulatory subunits should unravel the details for a variety of transmembrane signalling reactions. Thus, at least for three basic transmembrane signalling mechanisms: ligand modulated ion transport; ligand-modulated receptor enzyme activity (e.g. tyrosine kinase); and ligand-modulated liberation of cryptic mediators (like the alpha- and beta-subunits of the guanine nucleotide regulatory complexes) one can look forward with excitement to the elucidation in the not-too-distant future of a number of specific biochemical reaction pathways that lead to cell activation.

Animals↗