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Agrin binds to the nerve-muscle basal lamina via laminin.

Denzer AJ, Brandenberger R, Gesemann M, Chiquet M, Ruegg MA - J. Cell Biol. (1997)

Bottom Line: After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood.In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment.These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Biozentrum, University of Basel, Switzerland.

ABSTRACT
Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

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Binding of agrin to laminin-1 is of high affinity. Doseresponse curve of the binding of 125I-cAgrin7 to laminin-1. Halfmaximal binding was reached at ∼5 nM, suggesting a rather  strong binding of agrin to laminin-1. The binding curve shown results from one representative experiment. Background values  (BSA-coated wells) are subtracted from each data point. Values  shown are the mean ± SEM from three measurements.
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Figure 4: Binding of agrin to laminin-1 is of high affinity. Doseresponse curve of the binding of 125I-cAgrin7 to laminin-1. Halfmaximal binding was reached at ∼5 nM, suggesting a rather strong binding of agrin to laminin-1. The binding curve shown results from one representative experiment. Background values (BSA-coated wells) are subtracted from each data point. Values shown are the mean ± SEM from three measurements.

Mentions: The strength of the binding of agrin to laminin-1 was measured by dose-response curves using 125I-cAgrin7. On immobilized laminin-1, half-maximal binding (EC50) was reached at ∼5 nM (Fig. 4), suggesting a rather strong interaction of agrin with laminin-1. This binding is of similar strength as the binding of laminin-1 to nidogen (Kd ∼1 nM; Fox et al., 1991). At 4 nM 125I-cAgrin7, 90.3 ± 0.5% (mean ± SEM; n = 3) of the binding was competed with 100 nM unlabeled cN257Fc, indicating that the majority of the binding of full-length agrin to laminin-1 is mediated by the NH2-terminal end. Since agrin is an HSPG (Denzer et al., 1995; Tsen et al., 1995a) and laminin-1 is known to bind to heparin (Ott et al., 1982; Yurchenco et al., 1993), the residual binding of cAgrin7 to laminin-1 could be due to the binding of the HS-GAG side chains of agrin to the heparin-binding site of laminin-1. Indeed, only the HS-GAG side chain-carrying construct cAgrin7, but not cN257Fc, binds to the elastase fragment E3, which contains the major heparin-binding site of laminin-1 (Ott et al., 1982; data not shown). In summary, our data show that laminin-1, the laminin isoform present in Matrigel™, is a basement membrane binding partner for agrin. Furthermore, they show that the interaction is mediated by the NH2-terminal end of agrin, and we therefore propose that this part of agrin forms a structural unit (see also Discussion).


Agrin binds to the nerve-muscle basal lamina via laminin.

Denzer AJ, Brandenberger R, Gesemann M, Chiquet M, Ruegg MA - J. Cell Biol. (1997)

Binding of agrin to laminin-1 is of high affinity. Doseresponse curve of the binding of 125I-cAgrin7 to laminin-1. Halfmaximal binding was reached at ∼5 nM, suggesting a rather  strong binding of agrin to laminin-1. The binding curve shown results from one representative experiment. Background values  (BSA-coated wells) are subtracted from each data point. Values  shown are the mean ± SEM from three measurements.
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Related In: Results  -  Collection

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Figure 4: Binding of agrin to laminin-1 is of high affinity. Doseresponse curve of the binding of 125I-cAgrin7 to laminin-1. Halfmaximal binding was reached at ∼5 nM, suggesting a rather strong binding of agrin to laminin-1. The binding curve shown results from one representative experiment. Background values (BSA-coated wells) are subtracted from each data point. Values shown are the mean ± SEM from three measurements.
Mentions: The strength of the binding of agrin to laminin-1 was measured by dose-response curves using 125I-cAgrin7. On immobilized laminin-1, half-maximal binding (EC50) was reached at ∼5 nM (Fig. 4), suggesting a rather strong interaction of agrin with laminin-1. This binding is of similar strength as the binding of laminin-1 to nidogen (Kd ∼1 nM; Fox et al., 1991). At 4 nM 125I-cAgrin7, 90.3 ± 0.5% (mean ± SEM; n = 3) of the binding was competed with 100 nM unlabeled cN257Fc, indicating that the majority of the binding of full-length agrin to laminin-1 is mediated by the NH2-terminal end. Since agrin is an HSPG (Denzer et al., 1995; Tsen et al., 1995a) and laminin-1 is known to bind to heparin (Ott et al., 1982; Yurchenco et al., 1993), the residual binding of cAgrin7 to laminin-1 could be due to the binding of the HS-GAG side chains of agrin to the heparin-binding site of laminin-1. Indeed, only the HS-GAG side chain-carrying construct cAgrin7, but not cN257Fc, binds to the elastase fragment E3, which contains the major heparin-binding site of laminin-1 (Ott et al., 1982; data not shown). In summary, our data show that laminin-1, the laminin isoform present in Matrigel™, is a basement membrane binding partner for agrin. Furthermore, they show that the interaction is mediated by the NH2-terminal end of agrin, and we therefore propose that this part of agrin forms a structural unit (see also Discussion).

Bottom Line: After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood.In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment.These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Biozentrum, University of Basel, Switzerland.

ABSTRACT
Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

Show MeSH
Related in: MedlinePlus