Limits...
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

Related in: MedlinePlus

Chick agrin synthesized in vivo  contains the laminin-binding domain. (a)  Western blot of recombinant cAgrin7 and  agrin purified from vitreous fluid (VF agrin).  Transferred protein were stained with antiagrin antibodies (α agrin) or antilaminin antibodies (α laminin). Agrin purified from vitreous fluid has approximately the same  apparent molecular mass as recombinant  full-length agrin. The band detected at ∼115  kD most likely results from proteolytic degradation. No laminin-like immunoreactivity  is associated with recombinant agrin (right).  In contrast to this, the agrin-containing fractions from vitreous fluid are positive for  laminin-like protein. Based on their apparent molecular mass and the specificity of the  antiserum used (Brubacher et al., 1991),  they may represent nidogen (∼150 kD), the  β and γ chain (200 kD), and α chain (400  kD). Molecular masses of standard proteins  are given in kD. (b) cAgrin7 and agrin from  vitreous fluid were bound to immobilized  antiagrin mAb 5B1 and incubated with 125I– laminin-1. The data result from one representative experiment and are the mean ± SD of three measurements. Background counts (no agrin added; 132 ± 15 cpm) was subtracted. 100 nM cN257Fc (+ cN257Fc) inhibits binding of 125I–laminin-1 to cAgrin7 and VF agrin by more than 97%, indicating that the  NH2-terminal domain is responsible for the binding.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139873&req=5

Figure 5: Chick agrin synthesized in vivo contains the laminin-binding domain. (a) Western blot of recombinant cAgrin7 and agrin purified from vitreous fluid (VF agrin). Transferred protein were stained with antiagrin antibodies (α agrin) or antilaminin antibodies (α laminin). Agrin purified from vitreous fluid has approximately the same apparent molecular mass as recombinant full-length agrin. The band detected at ∼115 kD most likely results from proteolytic degradation. No laminin-like immunoreactivity is associated with recombinant agrin (right). In contrast to this, the agrin-containing fractions from vitreous fluid are positive for laminin-like protein. Based on their apparent molecular mass and the specificity of the antiserum used (Brubacher et al., 1991), they may represent nidogen (∼150 kD), the β and γ chain (200 kD), and α chain (400 kD). Molecular masses of standard proteins are given in kD. (b) cAgrin7 and agrin from vitreous fluid were bound to immobilized antiagrin mAb 5B1 and incubated with 125I– laminin-1. The data result from one representative experiment and are the mean ± SD of three measurements. Background counts (no agrin added; 132 ± 15 cpm) was subtracted. 100 nM cN257Fc (+ cN257Fc) inhibits binding of 125I–laminin-1 to cAgrin7 and VF agrin by more than 97%, indicating that the NH2-terminal domain is responsible for the binding.

Mentions: The results presented so far show that recombinant chick agrin, expressed in mammalian cells, binds to laminin-1. If this binding were important for agrin's function in vivo, agrin purified from tissue should have the same binding properties. To test this, agrin was isolated by anion exchange chromatography from the vitreous fluid of embryonic day 14 chick eyes, a rich source for agrin and other molecules secreted from retinal cells (e.g., Ruegg et al., 1989; Halfter, 1993; Denzer et al., 1995). As a control, cAgrin7 from conditioned medium of stably transfected HEK 293 cells was purified by the same procedure. When agrin-containing fractions were assayed by Western blot, agrin-like protein from vitreous fluid and cAgrin7 had a high apparent molecular mass (Fig. 5 a, left), which is consistent with previous results (Denzer et al., 1995). The bands with an apparent molecular mass of ∼115 kD are probably due to proteolytic degradation similar to what has been reported elsewhere (e.g., Nitkin et al., 1987; Godfrey, 1991; Rupp et al., 1991; Denzer et al., 1995). The same samples were also probed with antiserum 245, raised against native chick heart laminin (Brubacher et al., 1991). No laminin-like immunoreactivity was observed in the samples containing cAgrin7, derived from stably transfected HEK 293 cells, whereas immunoreactive bands were visible in the sample containing agrin from the vitreous fluid (Fig. 5 a, right). The strong immunoreactive band with the apparent molecular mass of 200 kD most likely corresponds to the β/γ subunits of laminin isoforms, while the faint bands with the molecular masses of 400 and 150 kD could represent α subunits and nidogen, respectively (Brubacher et al., 1991). The copurification of chick laminin isoforms from the vitreous fluid with agrin by a procedure that is selective for highly charged anions is unexpected and may reflect the binding of laminin to agrin. Consistent with this idea, Halfter (1993) reported copurification of laminin with a chick HSPG, which later was shown to be agrin (Tsen et al., 1995a).


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

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

Chick agrin synthesized in vivo  contains the laminin-binding domain. (a)  Western blot of recombinant cAgrin7 and  agrin purified from vitreous fluid (VF agrin).  Transferred protein were stained with antiagrin antibodies (α agrin) or antilaminin antibodies (α laminin). Agrin purified from vitreous fluid has approximately the same  apparent molecular mass as recombinant  full-length agrin. The band detected at ∼115  kD most likely results from proteolytic degradation. No laminin-like immunoreactivity  is associated with recombinant agrin (right).  In contrast to this, the agrin-containing fractions from vitreous fluid are positive for  laminin-like protein. Based on their apparent molecular mass and the specificity of the  antiserum used (Brubacher et al., 1991),  they may represent nidogen (∼150 kD), the  β and γ chain (200 kD), and α chain (400  kD). Molecular masses of standard proteins  are given in kD. (b) cAgrin7 and agrin from  vitreous fluid were bound to immobilized  antiagrin mAb 5B1 and incubated with 125I– laminin-1. The data result from one representative experiment and are the mean ± SD of three measurements. Background counts (no agrin added; 132 ± 15 cpm) was subtracted. 100 nM cN257Fc (+ cN257Fc) inhibits binding of 125I–laminin-1 to cAgrin7 and VF agrin by more than 97%, indicating that the  NH2-terminal domain is responsible for the binding.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Chick agrin synthesized in vivo contains the laminin-binding domain. (a) Western blot of recombinant cAgrin7 and agrin purified from vitreous fluid (VF agrin). Transferred protein were stained with antiagrin antibodies (α agrin) or antilaminin antibodies (α laminin). Agrin purified from vitreous fluid has approximately the same apparent molecular mass as recombinant full-length agrin. The band detected at ∼115 kD most likely results from proteolytic degradation. No laminin-like immunoreactivity is associated with recombinant agrin (right). In contrast to this, the agrin-containing fractions from vitreous fluid are positive for laminin-like protein. Based on their apparent molecular mass and the specificity of the antiserum used (Brubacher et al., 1991), they may represent nidogen (∼150 kD), the β and γ chain (200 kD), and α chain (400 kD). Molecular masses of standard proteins are given in kD. (b) cAgrin7 and agrin from vitreous fluid were bound to immobilized antiagrin mAb 5B1 and incubated with 125I– laminin-1. The data result from one representative experiment and are the mean ± SD of three measurements. Background counts (no agrin added; 132 ± 15 cpm) was subtracted. 100 nM cN257Fc (+ cN257Fc) inhibits binding of 125I–laminin-1 to cAgrin7 and VF agrin by more than 97%, indicating that the NH2-terminal domain is responsible for the binding.
Mentions: The results presented so far show that recombinant chick agrin, expressed in mammalian cells, binds to laminin-1. If this binding were important for agrin's function in vivo, agrin purified from tissue should have the same binding properties. To test this, agrin was isolated by anion exchange chromatography from the vitreous fluid of embryonic day 14 chick eyes, a rich source for agrin and other molecules secreted from retinal cells (e.g., Ruegg et al., 1989; Halfter, 1993; Denzer et al., 1995). As a control, cAgrin7 from conditioned medium of stably transfected HEK 293 cells was purified by the same procedure. When agrin-containing fractions were assayed by Western blot, agrin-like protein from vitreous fluid and cAgrin7 had a high apparent molecular mass (Fig. 5 a, left), which is consistent with previous results (Denzer et al., 1995). The bands with an apparent molecular mass of ∼115 kD are probably due to proteolytic degradation similar to what has been reported elsewhere (e.g., Nitkin et al., 1987; Godfrey, 1991; Rupp et al., 1991; Denzer et al., 1995). The same samples were also probed with antiserum 245, raised against native chick heart laminin (Brubacher et al., 1991). No laminin-like immunoreactivity was observed in the samples containing cAgrin7, derived from stably transfected HEK 293 cells, whereas immunoreactive bands were visible in the sample containing agrin from the vitreous fluid (Fig. 5 a, right). The strong immunoreactive band with the apparent molecular mass of 200 kD most likely corresponds to the β/γ subunits of laminin isoforms, while the faint bands with the molecular masses of 400 and 150 kD could represent α subunits and nidogen, respectively (Brubacher et al., 1991). The copurification of chick laminin isoforms from the vitreous fluid with agrin by a procedure that is selective for highly charged anions is unexpected and may reflect the binding of laminin to agrin. Consistent with this idea, Halfter (1993) reported copurification of laminin with a chick HSPG, which later was shown to be agrin (Tsen et al., 1995a).

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