The small leucine-rich repeat proteoglycan biglycan binds to alpha-dystroglycan and is upregulated in dystrophic muscle.

The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface. The alpha-/beta-dystroglycan subcomplex of the DAPC forms a critical link between the cytoskeleton and the extracellular matrix. A ligand blot overlay assay was used to search for novel dystroglycan binding partners in postsynaptic membranes from Torpedo electric organ. An approximately 125-kD dystroglycan-binding polypeptide was purified and shown by peptide microsequencing to be the Torpedo ortholog of the small leucine-rich repeat chondroitin sulfate proteoglycan biglycan. Biglycan binding to alpha-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant alpha-dystroglycan. The biglycan binding site was mapped to the COOH-terminal third of alpha-dystroglycan. Glycosylation of alpha-dystroglycan is not necessary for this interaction, but binding is dependent upon the chondroitin sulfate side chains of biglycan. In muscle, biglycan is detected at both synaptic and nonsynaptic regions. Finally, biglycan expression is elevated in muscle from the dystrophic mdx mouse. These findings reveal a novel binding partner for alpha-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix. These results also raise the possibility of a role for biglycan in the pathogenesis, and perhaps the treatment, of muscular dystrophy.

Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan, and the cell surface.

Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with alpha-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner.

The alpha-dystroglycan-beta-dystroglycan complex. Membrane organization and relationship to an agrin receptor.

Aberrant expression of the dystrophin-associated protein complex is thought to underlie the pathogenesis of Duchenne dystrophy, Becker muscular dystrophy, and severe childhood autosomal recessive muscular dystrophy. Recently, our laboratory identified an agrin receptor from Torpedo electric organ postsynaptic membranes. It is a heteromer of 190- and 50-kDa subunits with similarity to two components of the dystrophin-associated protein complex of alpha- and beta-dystroglycan. We now confirm the relationship between the Torpedo agrin receptor and mammalian dystroglycans and provide further information about the structure of the alpha-dystroglycan-beta-dystroglycan complex. The sequences of three peptides from each Torpedo subunit were 69% identical to mammalian dystroglycans. An antiserum to mammalian beta-dystroglycan recognizes the Torpedo 50-kDa polypeptide. Additionally, like alpha-dystroglycan, the 190-kDa agrin receptor subunit binds laminin. Previous studies have indicated that alpha- and beta-dystroglycan arise by cleavage of a precursor protein. Tryptic peptide mapping of both subunits and amino-terminal sequencing of Torpedo beta-dystroglycan indicate a single cleavage site, corresponding to serine 654 of the mammalian dystroglycan precursor. Gel electrophoresis analysis indicates there is at least one intrachain disulfide bond in beta-dystroglycan. These results provide precise primary structures for alpha- and beta-dystroglycan.

Polyamines antagonize N-methyl-D-aspartate-evoked depolarizations, but reduce Mg2+ block.

This study utilized a grease-gap preparation to investigate the effects of polyamines on responses of CA1 hippocampal pyramidal cells to N-methyl-D-aspartate (NMDA) and on the block of the NMDA channel by Mg2+. In the absence of added Mg2+, 1,10-diaminodecane (0.1-1 mM) non-competitively antagonized NMDA-evoked depolarizations. Its antagonism slowly progressed to a stable value, was not use-dependent and did not reverse completely upon washout. Similar results were obtained with 100 microM spermine and 1 mM diethylenetriamine. Addition of 1 mM Mg2+ to the superfusion medium greatly reduced these effects. Conversely, the polyamines attenuated the blocking action of Mg2+. Postnatal treatment with alpha-difluoromethylornithine reduced the total polyamine content of area CA1 in 10- to 15-day-old rats almost to the adult level (although spermine content was unaffected). Mg2+ less potently antagonized NMDA-evoked depolarizations in slices from 10- to 15-day-old rats than in slices from adult rats, and this difference was unaffected by the alpha-difluoromethylornithine treatment. These results suggest (1) that there are rapid and slow components to the antagonism of NMDA-evoked depolarizations by polyamines, both of which may involve permeation of the polyamine into or through the NMDA channel: (2) that polyamine release in brain could modulate the Mg2+ sensitivity of responses to NMDA; and (3) that changes in the total content of endogenous polyamine do not explain developmental differences in the sensitivity of NMDA-evoked depolarizations to Mg2+.

Identification and purification of an agrin receptor from Torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans.

The selective concentration of neurotransmitter receptors at the postsynaptic membrane is an essential aspect of synaptic differentiation and function. Agrin is an extracellular matrix protein that is likely to direct the accumulation of acetylcholine receptors and several other postsynaptic elements at developing and regenerating neuromuscular junctions. How agrin interacts with the membrane to bring about these changes is unknown. We now report the identification and purification of a protein complex from Torpedo electric organ postsynaptic membranes that is likely to serve as an agrin receptor. The native receptor is a heteromeric complex of two membrane glycoproteins of 190 kDa and 50 kDa. The 190 kDa subunit is sufficient to bind ligand. Peptide sequence analysis revealed that the 190 kDa and 50 kDa subunits are related to the dystrophin-associated glycoproteins alpha- and beta-dystroglycan, respectively. No other candidate agrin receptors were detected. The identification of the agrin receptor opens new avenues toward a mechanistic understanding of synapse differentiation.

Autoreceptor regulation of glutamate and aspartate release from slices of the hippocampal CA1 area.

Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N-methyl-D-aspartate (NMDA)-receptor antagonists depressed the release of glutamate, aspartate, and gamma-aminobutyrate evoked by 50 mM K+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3-[(+/- )-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP) and D-2-amino-5-phosphonovalerate (D-AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 microM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D-AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K(+)-evoked release of aspartate. Conversely, 6-cyano-7-nitro-quinoxaline-2,3-dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.

Developmental increase in the sensitivity to magnesium of NMDA receptors on CA1 hippocampal pyramidal cells.

The N-methyl-D-aspartate (NMDA) receptor is involved in processes, such as associative learning, that are particularly important during early postnatal development. It has been suggested that the activity and regulation of this receptor changes during development. Activation of the NMDA receptor is normally limited by Mg2+ present in the extracellular fluid of brain. We have found that Mg2+ less potently antagonizes the depolarizing action of NMDA in developing rats than in adults. A grease-gap method was used to record depolarizations evoked in CA1 hippocampal pyramidal cells by the excitants NMDA and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate). In the adult CA1 area, Mg2+ shifted the NMDA concentration-response curve to the right in a manner consistent with voltage-dependent open channel block (uncompetitive antagonism) in a preparation with significant receptor reserve. The potency of Mg2+ increased during development; a greater than two-fold change in the EC50 for Mg2+ was observed between 10-15 days of age and adulthood. A concentration of 10 mM reduced the maximum response of CA1 pyramidal cells to NMDA in adult rats, but not in developing rats. In addition, Mg2+ often enhanced the maximum depolarizations evoked by NMDA in 10- to 15-day-old rats, but very seldom in adults. No significant developmental changes in AMPA-induced depolarizations were observed in the presence or absence of Mg2+. These results suggest that synaptically released glutamate will readily activate NMDA receptors during early development and that its ability to do this declines with the maturation of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of glutamate and aspartate release from the Schaffer collaterals and other projections of CA3 hippocampal pyramidal cells.

Excitatory synaptic transmission in the CNS can be modulated by endogenous substances and metabolic states that alter release of the transmitter, usually glutamate and/or aspartate. To explore this issue, we have studied the release of endogenous glutamate and aspartate from synaptic terminals of the CA3-derived Schaffer collateral, commissural and ipsilateral associational fibers in slices of hippocampal area CA1. These terminals release glutamate and aspartate in about a 5:1 ratio. The release process is modulated by adenosine, by the transmitters themselves and by nerve terminal metabolism. Adenosine inhibits the release of both amino acids by acting upon an A1 receptor. The transmitters, once released, can regulate their further release by acting upon both an NMDA and a non-NMDA (quisqualate/kainate) receptor. Activation of the NMDA receptor enhances the release of both glutamate and aspartate, whereas activation of the non-NMDA receptor depresses the release of aspartate only. Superfusion of CA1 slices with a glucose-deficient medium increases the release of both amino acids and reduces the glutamate/aspartate ratio. These results have implications for the regulation of excitatory synaptic transmission in the CA1 area and for the mechanism of hypoglycemic damage to CA1 pyramidal cells.

Kindling, prenatal exposure to ethanol and postnatal development selectively alter responses of hippocampal pyramidal cells to NMDA.

Our studies suggest that treatments, such as kindling and exposure to ethanol in utero, which produce irreversible pathological changes in brain function also selectively alter neuronal responses to NMDA. We have identified increases in agonist potency, which may result from enhanced NMDA receptor expression, as well as both positive and negative modifications of Mg2+ regulation. There is no proof as yet that NMDA receptor plasticity accounts for the kindling phenomenon, for the cognitive deficits associated with fetal exposure to ethanol or for developmental events. However, all the effects we obtained are in the appropriate direction. The same functional parameters altered by kindling and prenatal exposure to ethanol are also modified during the normal development of the brain. It is tempting to speculate that these pathological stimuli produce their effects through the same mechanisms that would normally be activated by developmental signals.

A grease-gap method for studying the excitatory amino acid pharmacology of CA1 hippocampal pyramidal cells.

A grease-gap method for studying the pharmacology of CA1 hippocampal pyramidal cells was developed with use of rat hippocampal slices that included only area CA1 and the retrohippocampal area. These slices were transferred to a two-compartment superfusion chamber and the pyramidal cell bodies in area CA1 were separated from their axons in the subiculum with a grease barrier. The CA1 pyramidal cells were depolarized relative to their axons by superfusion with N-methyl-D-aspartate (NMDA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and L-glutamate. NMDA was unusually potent in the CA1-subiculum slice compared to other preparations. The NMDA receptor antagonists D(-)-2-amino-5-phosphonovalerate (D-AP5), phencyclidine and Mg2+ shifted the NMDA dose-response curve to the right in a parallel manner. Similarly, the quisqualate receptor antagonist pentobarbitone shifted the AMPA dose-response curve to the right. Schild plots for these antagonists had slopes insignificantly different from 1. These results are consistent with the presence of a substantial NMDA receptor reserve on CA1 pyramidal cells. They are also in line with the high density of excitatory amino acid receptors on CA1 hippocampal pyramidal cells and with the known pharmacological properties of these receptors. Grease-gap studies on the CA1-subiculum slice fill the need for a means of obtaining quantitative pharmacological data on CA1 pyramidal cells.