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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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The laminin-binding domain is present in mouse and  human agrin. Deduced amino acid sequences of the expressed sequence tags (Lennon et al., 1996) from mouse and human agrin  were aligned to the first 173 amino acids of chick agrin. In addition, the first 67 amino acids of rat agrin are shown. Amino acids  identical to chick agrin are denoted with dots. The small letters in  the chick sequence represent the proposed signal sequence (Denzer et al., 1995) with the initiator methionine (bold) and the proposed signal sequence cleavage site (*). Underlined amino acids  show the position of the tryptic peptide derived from a HSPG of  bovine kidney (Hagen et al., 1993). Homology between chick and  human starts after the signal sequence cleavage site of chick (Cys  26). Amino acids between Cys 26 and Glu 149 are almost 90%  identical between human, mouse, and chick. Note that the amino  acid sequences of mouse and human agrin that precede the proposed signal peptide cleavage site (*) are not homologous to  chick and consist mainly of hydrophobic amino acids. Thus, as in  chick these stretches might be part of a signal sequence (lowercase letters). The homology between chick and rat begins at Asp  157 immediately after the boundary where chick is alternatively  spliced. The human and mouse sequences lack the seven–amino  acid–long insert at this splice site (dashes). These sequence data  are available from GenBank/EMBL/DDBJ under accession number U84406 (human agrin) and U84407 (mouse agrin).
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Figure 6: The laminin-binding domain is present in mouse and human agrin. Deduced amino acid sequences of the expressed sequence tags (Lennon et al., 1996) from mouse and human agrin were aligned to the first 173 amino acids of chick agrin. In addition, the first 67 amino acids of rat agrin are shown. Amino acids identical to chick agrin are denoted with dots. The small letters in the chick sequence represent the proposed signal sequence (Denzer et al., 1995) with the initiator methionine (bold) and the proposed signal sequence cleavage site (*). Underlined amino acids show the position of the tryptic peptide derived from a HSPG of bovine kidney (Hagen et al., 1993). Homology between chick and human starts after the signal sequence cleavage site of chick (Cys 26). Amino acids between Cys 26 and Glu 149 are almost 90% identical between human, mouse, and chick. Note that the amino acid sequences of mouse and human agrin that precede the proposed signal peptide cleavage site (*) are not homologous to chick and consist mainly of hydrophobic amino acids. Thus, as in chick these stretches might be part of a signal sequence (lowercase letters). The homology between chick and rat begins at Asp 157 immediately after the boundary where chick is alternatively spliced. The human and mouse sequences lack the seven–amino acid–long insert at this splice site (dashes). These sequence data are available from GenBank/EMBL/DDBJ under accession number U84406 (human agrin) and U84407 (mouse agrin).

Mentions: The NH2-terminal region required for the binding of agrin to laminin-1 has so far only been described in chick (Denzer et al., 1995). Full-length cDNA encoding rat agrin lacks this region and instead, the first follistatin-like domain is preceded by a sequence that has been proposed to serve as signal sequence (Rupp et al., 1991). To see whether the NH2-terminal region of chick agrin is found in other species, we searched for homologous sequences using the BLAST algorithm (Altschul et al., 1990). Four expressed sequence tags, isolated from different tissues in mouse and in human (Lennon et al., 1996), closely matched this amino acid sequence. Sequencing of the clones confirmed that the deduced amino acid sequences of mouse and human agrin are highly homologous to each other and to chick agrin (Fig. 6). The homology starts at residue 26 of chick agrin, which corresponds to the predicted cleavage site for the signal sequence (Denzer et al., 1995). In the stretch from residue 26 to 149, 96% of the amino acids are identical between mouse and human, and 90% are identical between chick and the mammalian sequences (Fig. 6). This is by far the most highly conserved region in agrin (see also Tsim et al., 1992), suggesting that this domain may also confer binding to laminin in mammals.


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

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

The laminin-binding domain is present in mouse and  human agrin. Deduced amino acid sequences of the expressed sequence tags (Lennon et al., 1996) from mouse and human agrin  were aligned to the first 173 amino acids of chick agrin. In addition, the first 67 amino acids of rat agrin are shown. Amino acids  identical to chick agrin are denoted with dots. The small letters in  the chick sequence represent the proposed signal sequence (Denzer et al., 1995) with the initiator methionine (bold) and the proposed signal sequence cleavage site (*). Underlined amino acids  show the position of the tryptic peptide derived from a HSPG of  bovine kidney (Hagen et al., 1993). Homology between chick and  human starts after the signal sequence cleavage site of chick (Cys  26). Amino acids between Cys 26 and Glu 149 are almost 90%  identical between human, mouse, and chick. Note that the amino  acid sequences of mouse and human agrin that precede the proposed signal peptide cleavage site (*) are not homologous to  chick and consist mainly of hydrophobic amino acids. Thus, as in  chick these stretches might be part of a signal sequence (lowercase letters). The homology between chick and rat begins at Asp  157 immediately after the boundary where chick is alternatively  spliced. The human and mouse sequences lack the seven–amino  acid–long insert at this splice site (dashes). These sequence data  are available from GenBank/EMBL/DDBJ under accession number U84406 (human agrin) and U84407 (mouse agrin).
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Related In: Results  -  Collection

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Figure 6: The laminin-binding domain is present in mouse and human agrin. Deduced amino acid sequences of the expressed sequence tags (Lennon et al., 1996) from mouse and human agrin were aligned to the first 173 amino acids of chick agrin. In addition, the first 67 amino acids of rat agrin are shown. Amino acids identical to chick agrin are denoted with dots. The small letters in the chick sequence represent the proposed signal sequence (Denzer et al., 1995) with the initiator methionine (bold) and the proposed signal sequence cleavage site (*). Underlined amino acids show the position of the tryptic peptide derived from a HSPG of bovine kidney (Hagen et al., 1993). Homology between chick and human starts after the signal sequence cleavage site of chick (Cys 26). Amino acids between Cys 26 and Glu 149 are almost 90% identical between human, mouse, and chick. Note that the amino acid sequences of mouse and human agrin that precede the proposed signal peptide cleavage site (*) are not homologous to chick and consist mainly of hydrophobic amino acids. Thus, as in chick these stretches might be part of a signal sequence (lowercase letters). The homology between chick and rat begins at Asp 157 immediately after the boundary where chick is alternatively spliced. The human and mouse sequences lack the seven–amino acid–long insert at this splice site (dashes). These sequence data are available from GenBank/EMBL/DDBJ under accession number U84406 (human agrin) and U84407 (mouse agrin).
Mentions: The NH2-terminal region required for the binding of agrin to laminin-1 has so far only been described in chick (Denzer et al., 1995). Full-length cDNA encoding rat agrin lacks this region and instead, the first follistatin-like domain is preceded by a sequence that has been proposed to serve as signal sequence (Rupp et al., 1991). To see whether the NH2-terminal region of chick agrin is found in other species, we searched for homologous sequences using the BLAST algorithm (Altschul et al., 1990). Four expressed sequence tags, isolated from different tissues in mouse and in human (Lennon et al., 1996), closely matched this amino acid sequence. Sequencing of the clones confirmed that the deduced amino acid sequences of mouse and human agrin are highly homologous to each other and to chick agrin (Fig. 6). The homology starts at residue 26 of chick agrin, which corresponds to the predicted cleavage site for the signal sequence (Denzer et al., 1995). In the stretch from residue 26 to 149, 96% of the amino acids are identical between mouse and human, and 90% are identical between chick and the mammalian sequences (Fig. 6). This is by far the most highly conserved region in agrin (see also Tsim et al., 1992), suggesting that this domain may also confer binding to laminin in mammals.

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