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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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Anti-α7 antibody induces AChR clustering in the absence of exogenous agrin or laminin. Purified H36-α7 antibody  was added to cultures of α7BX2-transfected C2C12 myofibers.  Rhodamine–bungarotoxin was used to identify AChR clusters 18 h  later. Whereas high concentrations of anti-α7 antibody inhibit  laminin-induced AChR clustering (Fig. 5) and colocalization  (Fig. 3), low concentrations of antibody cross-link the integrin,  promote its association with the cytoskeleton (Lowrey and Kaufman, 1989; Song et al., 1993), and induce AChR clustering.
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Figure 9: Anti-α7 antibody induces AChR clustering in the absence of exogenous agrin or laminin. Purified H36-α7 antibody was added to cultures of α7BX2-transfected C2C12 myofibers. Rhodamine–bungarotoxin was used to identify AChR clusters 18 h later. Whereas high concentrations of anti-α7 antibody inhibit laminin-induced AChR clustering (Fig. 5) and colocalization (Fig. 3), low concentrations of antibody cross-link the integrin, promote its association with the cytoskeleton (Lowrey and Kaufman, 1989; Song et al., 1993), and induce AChR clustering.

Mentions: Lastly, engaging the α7β1 integrin with laminin or cross-linking the exogenous integrin with anti-α7 antibody promotes a detergent-stable association of the integrin with the cell cytoskeleton (Lowrey and Kaufman, 1989; Song et al., 1993) and an increase in free intracellular calcium (Kwon, M.S., C.S. Park, K.R. Choi, S.J. Kaufman, and W.S. Song, manuscript in preparation). In the absence of added laminin or agrin, relatively low concentrations of anti-α7 antibody also promotes AChR clustering on α7BX2 transfected C2 myofibers (Fig. 9). At higher antibody concentrations, where excess antibody fails to cross-link the integrin, cluster formation is minimal. Thus, engaging the α7β1 integrin with either its ligand or with antibody is sufficient to promote AChR clustering.


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)

Anti-α7 antibody induces AChR clustering in the absence of exogenous agrin or laminin. Purified H36-α7 antibody  was added to cultures of α7BX2-transfected C2C12 myofibers.  Rhodamine–bungarotoxin was used to identify AChR clusters 18 h  later. Whereas high concentrations of anti-α7 antibody inhibit  laminin-induced AChR clustering (Fig. 5) and colocalization  (Fig. 3), low concentrations of antibody cross-link the integrin,  promote its association with the cytoskeleton (Lowrey and Kaufman, 1989; Song et al., 1993), and induce AChR clustering.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132957&req=5

Figure 9: Anti-α7 antibody induces AChR clustering in the absence of exogenous agrin or laminin. Purified H36-α7 antibody was added to cultures of α7BX2-transfected C2C12 myofibers. Rhodamine–bungarotoxin was used to identify AChR clusters 18 h later. Whereas high concentrations of anti-α7 antibody inhibit laminin-induced AChR clustering (Fig. 5) and colocalization (Fig. 3), low concentrations of antibody cross-link the integrin, promote its association with the cytoskeleton (Lowrey and Kaufman, 1989; Song et al., 1993), and induce AChR clustering.
Mentions: Lastly, engaging the α7β1 integrin with laminin or cross-linking the exogenous integrin with anti-α7 antibody promotes a detergent-stable association of the integrin with the cell cytoskeleton (Lowrey and Kaufman, 1989; Song et al., 1993) and an increase in free intracellular calcium (Kwon, M.S., C.S. Park, K.R. Choi, S.J. Kaufman, and W.S. Song, manuscript in preparation). In the absence of added laminin or agrin, relatively low concentrations of anti-α7 antibody also promotes AChR clustering on α7BX2 transfected C2 myofibers (Fig. 9). At higher antibody concentrations, where excess antibody fails to cross-link the integrin, cluster formation is minimal. Thus, engaging the α7β1 integrin with either its ligand or with antibody is sufficient to promote AChR clustering.

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