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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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Agrin binds to  laminin isoforms expressed  in muscle. (a) Immunoblot  using antiagrin antibodies of  laminin isoforms purified  from EDTA extracts of adult  chick heart by immunoaffinity column with the anti-γ1  subunit–specific mAb 11B7  (Brandenberger and Chiquet, 1995). Agrin-like protein with the same apparent  molecular mass as cAgrin7 is  detected in the laminin preparation. Since no α-dystroglycan was detected in the  same laminin preparation  (data not shown), the copurification of agrin suggests that  laminins are associated with  agrin in muscle tissue. (b and  c) Agrin binds directly to purified chick laminin-2 and -4.  Equal amounts of the two  laminins were immobilized on microtiter plates and incubated either with 5 nM of iodinated cAgrin7 (b) or cN257Fc (c). Values given  are the result of one representative experiment and represent the mean ± SD of three measurements where the background (BSAcoated wells) has been subtracted. Background values were 163 ± 24 cpm in b and 75 ± 11 cpm in c. The binding of cAgrin7 to laminin-4 is  fivefold stronger than to laminin-2 (b) and a similar difference in the binding (fourfold) is observed with cN257Fc (c). Note that the difference in the counts measured with 125I-cAgrin7 and 125I-cN257Fc is due to different iodination efficiencies.
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Figure 7: Agrin binds to laminin isoforms expressed in muscle. (a) Immunoblot using antiagrin antibodies of laminin isoforms purified from EDTA extracts of adult chick heart by immunoaffinity column with the anti-γ1 subunit–specific mAb 11B7 (Brandenberger and Chiquet, 1995). Agrin-like protein with the same apparent molecular mass as cAgrin7 is detected in the laminin preparation. Since no α-dystroglycan was detected in the same laminin preparation (data not shown), the copurification of agrin suggests that laminins are associated with agrin in muscle tissue. (b and c) Agrin binds directly to purified chick laminin-2 and -4. Equal amounts of the two laminins were immobilized on microtiter plates and incubated either with 5 nM of iodinated cAgrin7 (b) or cN257Fc (c). Values given are the result of one representative experiment and represent the mean ± SD of three measurements where the background (BSAcoated wells) has been subtracted. Background values were 163 ± 24 cpm in b and 75 ± 11 cpm in c. The binding of cAgrin7 to laminin-4 is fivefold stronger than to laminin-2 (b) and a similar difference in the binding (fourfold) is observed with cN257Fc (c). Note that the difference in the counts measured with 125I-cAgrin7 and 125I-cN257Fc is due to different iodination efficiencies.

Mentions: Since we were interested in whether agrin's NH2-terminal domain would also mediate the tethering to muscle cell basal lamina, we measured binding of cAgrin7 and cN257Fc to laminin-2 and -4. The source for laminin-2 and -4 was a laminin preparation isolated from adult chick heart after EDTA extraction and sequential purification by wheat germ agglutinin Sepharose and immunoaffinity chromatography (Brandenberger and Chiquet, 1995). As shown by Western blot analysis using antiagrin antibodies, this laminin preparation contained substantial amounts of agrinlike protein (Fig. 7 a). As this laminin preparation did not contain α-dystroglycan, detected by a transfer overlay assay using iodinated chick agrin (data not shown), we conclude that the copurification of agrin with laminin isoforms from cardiac muscle is most likely a consequence of the association of agrin with the laminins. To test directly whether laminin-2 and -4 are binding proteins for agrin, we performed a solid-phase radioligand binding assay using purified laminin isoforms. With the same amount of laminin-2 and -4 coated on the microtiter wells, 5 nM of 125I-cAgrin7 gave a clear signal on laminin-2 and a fivefold stronger signal on laminin-4 (Fig. 7 b). Like cAgrin7, the NH2-terminal fragment, cN257Fc, also bound approximately four times more strongly to laminin-4 than to laminin-2 (Fig. 7 c). The weaker signal on laminin-2 was not due to a contamination with laminin-4, since this isoform represents less than 5% in the laminin-2 preparation (Brandenberger et al., 1996). A difference in the coating efficiency between laminin-2 and -4 was also excluded because equal amounts were immobilized when tested with an mAb specific for the α2 chain (Brandenberger et al., 1996). In summary, full-length chick agrin binds to both laminin-2 and -4, but at this particular concentration the interaction with the extrasynaptic laminin-2 is of lower apparent affinity.


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

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

Agrin binds to  laminin isoforms expressed  in muscle. (a) Immunoblot  using antiagrin antibodies of  laminin isoforms purified  from EDTA extracts of adult  chick heart by immunoaffinity column with the anti-γ1  subunit–specific mAb 11B7  (Brandenberger and Chiquet, 1995). Agrin-like protein with the same apparent  molecular mass as cAgrin7 is  detected in the laminin preparation. Since no α-dystroglycan was detected in the  same laminin preparation  (data not shown), the copurification of agrin suggests that  laminins are associated with  agrin in muscle tissue. (b and  c) Agrin binds directly to purified chick laminin-2 and -4.  Equal amounts of the two  laminins were immobilized on microtiter plates and incubated either with 5 nM of iodinated cAgrin7 (b) or cN257Fc (c). Values given  are the result of one representative experiment and represent the mean ± SD of three measurements where the background (BSAcoated wells) has been subtracted. Background values were 163 ± 24 cpm in b and 75 ± 11 cpm in c. The binding of cAgrin7 to laminin-4 is  fivefold stronger than to laminin-2 (b) and a similar difference in the binding (fourfold) is observed with cN257Fc (c). Note that the difference in the counts measured with 125I-cAgrin7 and 125I-cN257Fc is due to different iodination efficiencies.
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Figure 7: Agrin binds to laminin isoforms expressed in muscle. (a) Immunoblot using antiagrin antibodies of laminin isoforms purified from EDTA extracts of adult chick heart by immunoaffinity column with the anti-γ1 subunit–specific mAb 11B7 (Brandenberger and Chiquet, 1995). Agrin-like protein with the same apparent molecular mass as cAgrin7 is detected in the laminin preparation. Since no α-dystroglycan was detected in the same laminin preparation (data not shown), the copurification of agrin suggests that laminins are associated with agrin in muscle tissue. (b and c) Agrin binds directly to purified chick laminin-2 and -4. Equal amounts of the two laminins were immobilized on microtiter plates and incubated either with 5 nM of iodinated cAgrin7 (b) or cN257Fc (c). Values given are the result of one representative experiment and represent the mean ± SD of three measurements where the background (BSAcoated wells) has been subtracted. Background values were 163 ± 24 cpm in b and 75 ± 11 cpm in c. The binding of cAgrin7 to laminin-4 is fivefold stronger than to laminin-2 (b) and a similar difference in the binding (fourfold) is observed with cN257Fc (c). Note that the difference in the counts measured with 125I-cAgrin7 and 125I-cN257Fc is due to different iodination efficiencies.
Mentions: Since we were interested in whether agrin's NH2-terminal domain would also mediate the tethering to muscle cell basal lamina, we measured binding of cAgrin7 and cN257Fc to laminin-2 and -4. The source for laminin-2 and -4 was a laminin preparation isolated from adult chick heart after EDTA extraction and sequential purification by wheat germ agglutinin Sepharose and immunoaffinity chromatography (Brandenberger and Chiquet, 1995). As shown by Western blot analysis using antiagrin antibodies, this laminin preparation contained substantial amounts of agrinlike protein (Fig. 7 a). As this laminin preparation did not contain α-dystroglycan, detected by a transfer overlay assay using iodinated chick agrin (data not shown), we conclude that the copurification of agrin with laminin isoforms from cardiac muscle is most likely a consequence of the association of agrin with the laminins. To test directly whether laminin-2 and -4 are binding proteins for agrin, we performed a solid-phase radioligand binding assay using purified laminin isoforms. With the same amount of laminin-2 and -4 coated on the microtiter wells, 5 nM of 125I-cAgrin7 gave a clear signal on laminin-2 and a fivefold stronger signal on laminin-4 (Fig. 7 b). Like cAgrin7, the NH2-terminal fragment, cN257Fc, also bound approximately four times more strongly to laminin-4 than to laminin-2 (Fig. 7 c). The weaker signal on laminin-2 was not due to a contamination with laminin-4, since this isoform represents less than 5% in the laminin-2 preparation (Brandenberger et al., 1996). A difference in the coating efficiency between laminin-2 and -4 was also excluded because equal amounts were immobilized when tested with an mAb specific for the α2 chain (Brandenberger et al., 1996). In summary, full-length chick agrin binds to both laminin-2 and -4, but at this particular concentration the interaction with the extrasynaptic laminin-2 is of lower apparent affinity.

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