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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-1. Solid-phase radioligand binding assay with  purified proteins. Mouse  laminin-1, nidogen, and chick  α-dystroglycan were immobilized in microtiter plates and  subsequently incubated with  125I-cAgrin7 or 125I-cN257Fc.  Each value represents binding after subtraction of the  background (BSA-coated  wells). The values given are  the mean ± SD from one  representative experiment  with three independent measurements in the case of 125IcN257Fc and two measurements for 125I-cAgrin7. (a) 5  nM of 125I-cAgrin7 bound to α-dystroglycan and to laminin-1, but not to nidogen. Background counts were 89 ± 15 cpm. (b) 5 nM 125IcN257Fc bound to laminin-1 but not to α-dystroglycan and nidogen. Background counts were 65 ± 3 cpm. These experiments show that  the binding of agrin to laminin is mediated by the NH2-terminal end of agrin. Counts measured in a and b cannot be compared because  cAgrin7 and cN257Fc are iodinated to different extents.
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Figure 3: Agrin binds to laminin-1. Solid-phase radioligand binding assay with purified proteins. Mouse laminin-1, nidogen, and chick α-dystroglycan were immobilized in microtiter plates and subsequently incubated with 125I-cAgrin7 or 125I-cN257Fc. Each value represents binding after subtraction of the background (BSA-coated wells). The values given are the mean ± SD from one representative experiment with three independent measurements in the case of 125IcN257Fc and two measurements for 125I-cAgrin7. (a) 5 nM of 125I-cAgrin7 bound to α-dystroglycan and to laminin-1, but not to nidogen. Background counts were 89 ± 15 cpm. (b) 5 nM 125IcN257Fc bound to laminin-1 but not to α-dystroglycan and nidogen. Background counts were 65 ± 3 cpm. These experiments show that the binding of agrin to laminin is mediated by the NH2-terminal end of agrin. Counts measured in a and b cannot be compared because cAgrin7 and cN257Fc are iodinated to different extents.

Mentions: Matrigel™ consists of a mixture of several ECM molecules, such as collagen type IV, laminin-1, nidogen/entactin, and perlecan (Kleinman et al., 1982). To identify the components to which agrin binds, a solid-phase radioligand binding assay was used (Gesemann et al., 1996). Laminin-1 and perlecan were purified from the Engelbreth-HolmSwarm mouse tumor (Timpl et al., 1979), and collagen type IV was isolated from human placenta (Weber et al., 1984; Ries et al., 1995). After coating the purified proteins onto the plastic surface of a microtiter plate, wells were incubated with 5 nM 125I-cAgrin7 or 125I-cN257Fc and bound radioactivity was measured. Both agrin constructs bound to laminin-1 (Fig. 3), while no or only little binding was seen to perlecan and collagen type IV (data not shown). Since laminins bind to nidogen with high affinity (Kd ∼1 nM; Fox et al., 1991), most laminin preparations also contain nidogen (Paulsson et al., 1987). To test whether the binding of agrin to laminin-1 was due to nidogen, we also measured binding of cAgrin7 and cN257Fc to recombinant mouse nidogen, expressed in stably transfected HEK 293 cells (Fox et al., 1991). No binding was observed for both agrin constructs (Fig. 3). Hence, the binding of agrin to the laminin–nidogen complex is based on a direct interaction with laminin-1. As expected from previous experiments (Bowe et al., 1994; Campanelli et al., 1994; Gee et al., 1994; Sugiyama et al., 1994; Gesemann et al., 1996), cAgrin7 also bound to α-dystroglycan, purified from chick skeletal muscle (Brancaccio et al., 1995; Fig. 3 a). In contrast, the NH2terminal fragment, cN257Fc, did not bind (Fig. 3 b). This is consistent with the notion that the binding site to this peripheral membrane protein resides in its COOH-terminal half of agrin (Bowe et al., 1994; Campanelli et al., 1994; Gee et al., 1994; Sugiyama et al., 1994; Gesemann et al., 1996). Accordingly, only the laminin-binding site is common to cN257Fc and cAgrin7. In addition, no binding of 125I-r21Fc was observed to any of the components tested (data not shown), which excludes a contribution of the Fc part in the binding of cN257Fc to laminin-1.


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-1. Solid-phase radioligand binding assay with  purified proteins. Mouse  laminin-1, nidogen, and chick  α-dystroglycan were immobilized in microtiter plates and  subsequently incubated with  125I-cAgrin7 or 125I-cN257Fc.  Each value represents binding after subtraction of the  background (BSA-coated  wells). The values given are  the mean ± SD from one  representative experiment  with three independent measurements in the case of 125IcN257Fc and two measurements for 125I-cAgrin7. (a) 5  nM of 125I-cAgrin7 bound to α-dystroglycan and to laminin-1, but not to nidogen. Background counts were 89 ± 15 cpm. (b) 5 nM 125IcN257Fc bound to laminin-1 but not to α-dystroglycan and nidogen. Background counts were 65 ± 3 cpm. These experiments show that  the binding of agrin to laminin is mediated by the NH2-terminal end of agrin. Counts measured in a and b cannot be compared because  cAgrin7 and cN257Fc are iodinated to different extents.
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Related In: Results  -  Collection

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Figure 3: Agrin binds to laminin-1. Solid-phase radioligand binding assay with purified proteins. Mouse laminin-1, nidogen, and chick α-dystroglycan were immobilized in microtiter plates and subsequently incubated with 125I-cAgrin7 or 125I-cN257Fc. Each value represents binding after subtraction of the background (BSA-coated wells). The values given are the mean ± SD from one representative experiment with three independent measurements in the case of 125IcN257Fc and two measurements for 125I-cAgrin7. (a) 5 nM of 125I-cAgrin7 bound to α-dystroglycan and to laminin-1, but not to nidogen. Background counts were 89 ± 15 cpm. (b) 5 nM 125IcN257Fc bound to laminin-1 but not to α-dystroglycan and nidogen. Background counts were 65 ± 3 cpm. These experiments show that the binding of agrin to laminin is mediated by the NH2-terminal end of agrin. Counts measured in a and b cannot be compared because cAgrin7 and cN257Fc are iodinated to different extents.
Mentions: Matrigel™ consists of a mixture of several ECM molecules, such as collagen type IV, laminin-1, nidogen/entactin, and perlecan (Kleinman et al., 1982). To identify the components to which agrin binds, a solid-phase radioligand binding assay was used (Gesemann et al., 1996). Laminin-1 and perlecan were purified from the Engelbreth-HolmSwarm mouse tumor (Timpl et al., 1979), and collagen type IV was isolated from human placenta (Weber et al., 1984; Ries et al., 1995). After coating the purified proteins onto the plastic surface of a microtiter plate, wells were incubated with 5 nM 125I-cAgrin7 or 125I-cN257Fc and bound radioactivity was measured. Both agrin constructs bound to laminin-1 (Fig. 3), while no or only little binding was seen to perlecan and collagen type IV (data not shown). Since laminins bind to nidogen with high affinity (Kd ∼1 nM; Fox et al., 1991), most laminin preparations also contain nidogen (Paulsson et al., 1987). To test whether the binding of agrin to laminin-1 was due to nidogen, we also measured binding of cAgrin7 and cN257Fc to recombinant mouse nidogen, expressed in stably transfected HEK 293 cells (Fox et al., 1991). No binding was observed for both agrin constructs (Fig. 3). Hence, the binding of agrin to the laminin–nidogen complex is based on a direct interaction with laminin-1. As expected from previous experiments (Bowe et al., 1994; Campanelli et al., 1994; Gee et al., 1994; Sugiyama et al., 1994; Gesemann et al., 1996), cAgrin7 also bound to α-dystroglycan, purified from chick skeletal muscle (Brancaccio et al., 1995; Fig. 3 a). In contrast, the NH2terminal fragment, cN257Fc, did not bind (Fig. 3 b). This is consistent with the notion that the binding site to this peripheral membrane protein resides in its COOH-terminal half of agrin (Bowe et al., 1994; Campanelli et al., 1994; Gee et al., 1994; Sugiyama et al., 1994; Gesemann et al., 1996). Accordingly, only the laminin-binding site is common to cN257Fc and cAgrin7. In addition, no binding of 125I-r21Fc was observed to any of the components tested (data not shown), which excludes a contribution of the Fc part in the binding of cN257Fc to laminin-1.

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