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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.

Show MeSH
Structure and biochemical analysis of agrin constructs. (a) Structural organization of chick agrin and of fragments used in this  study. Symbols and designations of individual domains are according to Bork and Bairoch (1995. Trends Biochem. Sci. 20): FS, follistatin-like module; EG, EGF-like module; LE, laminin EGF-like module; SEA, module first found in sea urchin sperm protein, enterokinase  and agrin; LamG, laminin G–like module. Furthermore, the fragment of the constant region of mouse immunoglobulin gamma heavy  chain (Fc), the chick agrin signal sequence (SS), the hemagglutinin signal sequence (SH), the serine/threonine rich regions (S/T), potential N-linked glycosylation sites, conserved GAG side chain attachment sites, and the sites of alternative mRNA splicing are indicated.  The NH2-terminal region of agrin characterized in this study is named NtA domain. Notes: (1) Inserts at splice sites A and B are not  specified. They are mentioned in the text if relevant for the experiment; and (2) the construct cΔN15Agrin was previously called cFull  (Denzer et al., 1995; Gesemann et al., 1995) and covers the coding region of chick agrin as described by Tsim et al. (1992). (b) Autoradiogram of the 35S-labeled agrin fragments depicted in a after precipitation from conditioned medium of transiently transfected COS  cells. cAgrin7 and cΔN15Agrin were immunoprecipitated with anti-cΔN15Agrin antibodies (Denzer et al., 1995). cN257Fc and r21Fc were  precipitated with protein A–Sepharose. Proteins were separated by 3–12% SDS-PAGE. The two protein bands of cN257Fc are probably  due to inefficient stop of protein translation at the COOH-terminal end of the construct. Molecular masses in kD of standard proteins  are indicated.
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Figure 1: Structure and biochemical analysis of agrin constructs. (a) Structural organization of chick agrin and of fragments used in this study. Symbols and designations of individual domains are according to Bork and Bairoch (1995. Trends Biochem. Sci. 20): FS, follistatin-like module; EG, EGF-like module; LE, laminin EGF-like module; SEA, module first found in sea urchin sperm protein, enterokinase and agrin; LamG, laminin G–like module. Furthermore, the fragment of the constant region of mouse immunoglobulin gamma heavy chain (Fc), the chick agrin signal sequence (SS), the hemagglutinin signal sequence (SH), the serine/threonine rich regions (S/T), potential N-linked glycosylation sites, conserved GAG side chain attachment sites, and the sites of alternative mRNA splicing are indicated. The NH2-terminal region of agrin characterized in this study is named NtA domain. Notes: (1) Inserts at splice sites A and B are not specified. They are mentioned in the text if relevant for the experiment; and (2) the construct cΔN15Agrin was previously called cFull (Denzer et al., 1995; Gesemann et al., 1995) and covers the coding region of chick agrin as described by Tsim et al. (1992). (b) Autoradiogram of the 35S-labeled agrin fragments depicted in a after precipitation from conditioned medium of transiently transfected COS cells. cAgrin7 and cΔN15Agrin were immunoprecipitated with anti-cΔN15Agrin antibodies (Denzer et al., 1995). cN257Fc and r21Fc were precipitated with protein A–Sepharose. Proteins were separated by 3–12% SDS-PAGE. The two protein bands of cN257Fc are probably due to inefficient stop of protein translation at the COOH-terminal end of the construct. Molecular masses in kD of standard proteins are indicated.

Mentions: Culturing of primary chick myotubes and transfections of COS-7 (Gluzman, 1981) or HEK 293 cells (Graham et al., 1977) were carried out as described by Gesemann et al. (1995). Iodination of purified protein was performed as described (Gesemann et al., 1996). 35S labeling, immunoprecipitation, and immunoblots were essentially done as described (Denzer et al., 1995). Recombinant cAgrin7 and cΔN15Agrin (see Fig. 1 a) in the supernatant of transiently transfected COS cells were immunoprecipitated with the antiserum raised against cΔN15Agrin (Denzer et al., 1995), whereas cN257Fc and r21Fc (see Fig. 1 a) were directly bound to protein A–Sepharose (Pharmacia, LKB Biotechnology Inc., Piscataway, NJ). 35Slabeled proteins were analyzed by SDS-PAGE on a 3–12% gradient gel followed by fluorography. For immunoblots, 3 μg of purified cAgrin7 or agrin purified from vitreous fluid (VF agrin) and 5 μg of total chick heart laminins (Brandenberger and Chiquet, 1995) was separated by SDSPAGE on a 3–12% gradient gel, transferred to nitrocellulose membrane, and analyzed as described in Denzer et al. (1995).


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

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

Structure and biochemical analysis of agrin constructs. (a) Structural organization of chick agrin and of fragments used in this  study. Symbols and designations of individual domains are according to Bork and Bairoch (1995. Trends Biochem. Sci. 20): FS, follistatin-like module; EG, EGF-like module; LE, laminin EGF-like module; SEA, module first found in sea urchin sperm protein, enterokinase  and agrin; LamG, laminin G–like module. Furthermore, the fragment of the constant region of mouse immunoglobulin gamma heavy  chain (Fc), the chick agrin signal sequence (SS), the hemagglutinin signal sequence (SH), the serine/threonine rich regions (S/T), potential N-linked glycosylation sites, conserved GAG side chain attachment sites, and the sites of alternative mRNA splicing are indicated.  The NH2-terminal region of agrin characterized in this study is named NtA domain. Notes: (1) Inserts at splice sites A and B are not  specified. They are mentioned in the text if relevant for the experiment; and (2) the construct cΔN15Agrin was previously called cFull  (Denzer et al., 1995; Gesemann et al., 1995) and covers the coding region of chick agrin as described by Tsim et al. (1992). (b) Autoradiogram of the 35S-labeled agrin fragments depicted in a after precipitation from conditioned medium of transiently transfected COS  cells. cAgrin7 and cΔN15Agrin were immunoprecipitated with anti-cΔN15Agrin antibodies (Denzer et al., 1995). cN257Fc and r21Fc were  precipitated with protein A–Sepharose. Proteins were separated by 3–12% SDS-PAGE. The two protein bands of cN257Fc are probably  due to inefficient stop of protein translation at the COOH-terminal end of the construct. Molecular masses in kD of standard proteins  are indicated.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139873&req=5

Figure 1: Structure and biochemical analysis of agrin constructs. (a) Structural organization of chick agrin and of fragments used in this study. Symbols and designations of individual domains are according to Bork and Bairoch (1995. Trends Biochem. Sci. 20): FS, follistatin-like module; EG, EGF-like module; LE, laminin EGF-like module; SEA, module first found in sea urchin sperm protein, enterokinase and agrin; LamG, laminin G–like module. Furthermore, the fragment of the constant region of mouse immunoglobulin gamma heavy chain (Fc), the chick agrin signal sequence (SS), the hemagglutinin signal sequence (SH), the serine/threonine rich regions (S/T), potential N-linked glycosylation sites, conserved GAG side chain attachment sites, and the sites of alternative mRNA splicing are indicated. The NH2-terminal region of agrin characterized in this study is named NtA domain. Notes: (1) Inserts at splice sites A and B are not specified. They are mentioned in the text if relevant for the experiment; and (2) the construct cΔN15Agrin was previously called cFull (Denzer et al., 1995; Gesemann et al., 1995) and covers the coding region of chick agrin as described by Tsim et al. (1992). (b) Autoradiogram of the 35S-labeled agrin fragments depicted in a after precipitation from conditioned medium of transiently transfected COS cells. cAgrin7 and cΔN15Agrin were immunoprecipitated with anti-cΔN15Agrin antibodies (Denzer et al., 1995). cN257Fc and r21Fc were precipitated with protein A–Sepharose. Proteins were separated by 3–12% SDS-PAGE. The two protein bands of cN257Fc are probably due to inefficient stop of protein translation at the COOH-terminal end of the construct. Molecular masses in kD of standard proteins are indicated.
Mentions: Culturing of primary chick myotubes and transfections of COS-7 (Gluzman, 1981) or HEK 293 cells (Graham et al., 1977) were carried out as described by Gesemann et al. (1995). Iodination of purified protein was performed as described (Gesemann et al., 1996). 35S labeling, immunoprecipitation, and immunoblots were essentially done as described (Denzer et al., 1995). Recombinant cAgrin7 and cΔN15Agrin (see Fig. 1 a) in the supernatant of transiently transfected COS cells were immunoprecipitated with the antiserum raised against cΔN15Agrin (Denzer et al., 1995), whereas cN257Fc and r21Fc (see Fig. 1 a) were directly bound to protein A–Sepharose (Pharmacia, LKB Biotechnology Inc., Piscataway, NJ). 35Slabeled proteins were analyzed by SDS-PAGE on a 3–12% gradient gel followed by fluorography. For immunoblots, 3 μg of purified cAgrin7 or agrin purified from vitreous fluid (VF agrin) and 5 μg of total chick heart laminins (Brandenberger and Chiquet, 1995) was separated by SDSPAGE on a 3–12% gradient gel, transferred to nitrocellulose membrane, and analyzed as described in Denzer et al. (1995).

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