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A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering.

Burkin DJ, Gu M, Hodges BL, Campanelli JT, Kaufman SJ - J. Cell Biol. (1998)

Bottom Line: High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin.Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active.These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois 61801, USA.

ABSTRACT
The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.

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Coprecipitation of the AChR and α7β1 integrin. Anti-AChR antibody and protein G–agarose were used to immunoprecipitate extracts prepared from C2C12α7BX2 myotubes.  Western blot analysis using anti-α7 cytoplasmic domain B antibody of (1) uninduced cells, no anti-AChR antibody, (2) uninduced cells, (3) agrin-induced cells, (4) laminin and agrin-induced  cells, and (5) laminin-induced cells, reveals laminin-dependent  coprecipitation of the AChR and integrin. The α7 chain may undergo increased proteolytic processing (Song et al., 1992) in the  presence of both agrin and laminin.
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Figure 8: Coprecipitation of the AChR and α7β1 integrin. Anti-AChR antibody and protein G–agarose were used to immunoprecipitate extracts prepared from C2C12α7BX2 myotubes. Western blot analysis using anti-α7 cytoplasmic domain B antibody of (1) uninduced cells, no anti-AChR antibody, (2) uninduced cells, (3) agrin-induced cells, (4) laminin and agrin-induced cells, and (5) laminin-induced cells, reveals laminin-dependent coprecipitation of the AChR and integrin. The α7 chain may undergo increased proteolytic processing (Song et al., 1992) in the presence of both agrin and laminin.

Mentions: To further demonstrate a physiologic role for the α7β1 integrin in neuromuscular junction formation, immunoprecipitation was carried out to determine whether the AChR and integrin were physically associated. C2C12α7BX2 myotubes were induced with agrin, laminin, or agrin and laminin. Anti-AChR mAb and protein G–agarose were used to immunoprecipitate the AChR and its associated molecules. Western blot analysis reveals that the α7β1 integrin is coprecipitated with the AChR and that their association in this complex is dependent on induction with laminin. Whereas the integrin is not detected in uninduced fibers or upon induction with agrin, it is associated with AChR when laminin or laminin and agrin were used to promote clustering (Fig. 8). In the presence of both agrin and laminin, the α7 chain may undergo increased proteolytic processing (Song et al., 1992). This laminin-dependent association of the α7β1 integrin with AChRs further supports the conclusion that the integrin has a direct physiologic role in the clustering of AChR.


A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering.

Burkin DJ, Gu M, Hodges BL, Campanelli JT, Kaufman SJ - J. Cell Biol. (1998)

Coprecipitation of the AChR and α7β1 integrin. Anti-AChR antibody and protein G–agarose were used to immunoprecipitate extracts prepared from C2C12α7BX2 myotubes.  Western blot analysis using anti-α7 cytoplasmic domain B antibody of (1) uninduced cells, no anti-AChR antibody, (2) uninduced cells, (3) agrin-induced cells, (4) laminin and agrin-induced  cells, and (5) laminin-induced cells, reveals laminin-dependent  coprecipitation of the AChR and integrin. The α7 chain may undergo increased proteolytic processing (Song et al., 1992) in the  presence of both agrin and laminin.
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Related In: Results  -  Collection

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Figure 8: Coprecipitation of the AChR and α7β1 integrin. Anti-AChR antibody and protein G–agarose were used to immunoprecipitate extracts prepared from C2C12α7BX2 myotubes. Western blot analysis using anti-α7 cytoplasmic domain B antibody of (1) uninduced cells, no anti-AChR antibody, (2) uninduced cells, (3) agrin-induced cells, (4) laminin and agrin-induced cells, and (5) laminin-induced cells, reveals laminin-dependent coprecipitation of the AChR and integrin. The α7 chain may undergo increased proteolytic processing (Song et al., 1992) in the presence of both agrin and laminin.
Mentions: To further demonstrate a physiologic role for the α7β1 integrin in neuromuscular junction formation, immunoprecipitation was carried out to determine whether the AChR and integrin were physically associated. C2C12α7BX2 myotubes were induced with agrin, laminin, or agrin and laminin. Anti-AChR mAb and protein G–agarose were used to immunoprecipitate the AChR and its associated molecules. Western blot analysis reveals that the α7β1 integrin is coprecipitated with the AChR and that their association in this complex is dependent on induction with laminin. Whereas the integrin is not detected in uninduced fibers or upon induction with agrin, it is associated with AChR when laminin or laminin and agrin were used to promote clustering (Fig. 8). In the presence of both agrin and laminin, the α7 chain may undergo increased proteolytic processing (Song et al., 1992). This laminin-dependent association of the α7β1 integrin with AChRs further supports the conclusion that the integrin has a direct physiologic role in the clustering of AChR.

Bottom Line: High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin.Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active.These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois 61801, USA.

ABSTRACT
The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.

Show MeSH
Related in: MedlinePlus