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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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Antibody against endogenous α7β1 integrin inhibits  laminin-induced AChR clustering. AChR clustering was initiated  in RMo rat myotubes with agrin, laminin, or agrin and laminin.  Anti-α7 O26 antibody, reactive with the endogenous rat α7 integrin chain, had no effect on agrin-induced clustering (n = 55).  The antibody inhibited ∼50% of laminin-induce AChR clustering (n = 103) as well as the enhanced clustering promoted by  laminin and agrin (n = 154). n = number of clusters scored in  control wells (no antibody). The mean values scored in six samples per condition, ±SE, are given.
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Figure 5: Antibody against endogenous α7β1 integrin inhibits laminin-induced AChR clustering. AChR clustering was initiated in RMo rat myotubes with agrin, laminin, or agrin and laminin. Anti-α7 O26 antibody, reactive with the endogenous rat α7 integrin chain, had no effect on agrin-induced clustering (n = 55). The antibody inhibited ∼50% of laminin-induce AChR clustering (n = 103) as well as the enhanced clustering promoted by laminin and agrin (n = 154). n = number of clusters scored in control wells (no antibody). The mean values scored in six samples per condition, ±SE, are given.

Mentions: Because the anti-α7 mAbs react with rat but not mouse integrin, it was necessary to use rat myofibers to determine if the enhancement of AChR clustering fostered by induction with both laminin and agrin could be inhibited with anti-α7 antibody. Myofibers that developed from the RMo line of rat myoblasts were induced with agrin, laminin, or agrin and laminin. O26 anti-α7 antibody was added at the time of induction. In two separate experiments the antibody reduced the numbers of clusters to ∼50% that of control cultures induced with laminin. The number of AChR aggregates promoted by agrin was not diminished by antibody, whereas the enhancement of clustering promoted upon induction with both agrin and laminin was reduced to that promoted by agrin alone (Fig. 5).


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)

Antibody against endogenous α7β1 integrin inhibits  laminin-induced AChR clustering. AChR clustering was initiated  in RMo rat myotubes with agrin, laminin, or agrin and laminin.  Anti-α7 O26 antibody, reactive with the endogenous rat α7 integrin chain, had no effect on agrin-induced clustering (n = 55).  The antibody inhibited ∼50% of laminin-induce AChR clustering (n = 103) as well as the enhanced clustering promoted by  laminin and agrin (n = 154). n = number of clusters scored in  control wells (no antibody). The mean values scored in six samples per condition, ±SE, are given.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132957&req=5

Figure 5: Antibody against endogenous α7β1 integrin inhibits laminin-induced AChR clustering. AChR clustering was initiated in RMo rat myotubes with agrin, laminin, or agrin and laminin. Anti-α7 O26 antibody, reactive with the endogenous rat α7 integrin chain, had no effect on agrin-induced clustering (n = 55). The antibody inhibited ∼50% of laminin-induce AChR clustering (n = 103) as well as the enhanced clustering promoted by laminin and agrin (n = 154). n = number of clusters scored in control wells (no antibody). The mean values scored in six samples per condition, ±SE, are given.
Mentions: Because the anti-α7 mAbs react with rat but not mouse integrin, it was necessary to use rat myofibers to determine if the enhancement of AChR clustering fostered by induction with both laminin and agrin could be inhibited with anti-α7 antibody. Myofibers that developed from the RMo line of rat myoblasts were induced with agrin, laminin, or agrin and laminin. O26 anti-α7 antibody was added at the time of induction. In two separate experiments the antibody reduced the numbers of clusters to ∼50% that of control cultures induced with laminin. The number of AChR aggregates promoted by agrin was not diminished by antibody, whereas the enhancement of clustering promoted upon induction with both agrin and laminin was reduced to that promoted by agrin alone (Fig. 5).

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