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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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Cluster formation and colocalization of the α7 integrin 5 h after induction of transfected C2C12 myofibers with laminin, agrin,  or laminin and agrin. (A) The number of clusters that form in response to agrin and laminin exceeds that produced by either alone. (B)  Exogenous α7 does not localize well with agrin-induced clusters. In contrast, the synergistic response evoked by agrin and laminin is  characterized by localization of the α7β1 integrin with AChRs, even early in cluster formation. These results suggest that laminin, agrin  and the α7β1 integrin interact in a common or convergent pathway to form AChR clusters and neuromuscular junctions. The mean values scored in triplicate samples, ±SE, are given.
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Figure 6: Cluster formation and colocalization of the α7 integrin 5 h after induction of transfected C2C12 myofibers with laminin, agrin, or laminin and agrin. (A) The number of clusters that form in response to agrin and laminin exceeds that produced by either alone. (B) Exogenous α7 does not localize well with agrin-induced clusters. In contrast, the synergistic response evoked by agrin and laminin is characterized by localization of the α7β1 integrin with AChRs, even early in cluster formation. These results suggest that laminin, agrin and the α7β1 integrin interact in a common or convergent pathway to form AChR clusters and neuromuscular junctions. The mean values scored in triplicate samples, ±SE, are given.

Mentions: It was recently suggested that laminin and agrin initiate clustering by alternative pathways that can be distinguished both temporally and biochemically: agrin induces clustering more rapidly than laminin and requires MuSK (Sugiyama et al., 1997). We therefore examined early on in the induction process whether we could distinguish the association of the α7β1 integrin with agrin- and laminin-induced AChR clusters. Myofibers were treated with agrin, laminin, or with both agrin and laminin, and cluster formation was determined 5 h later. Initiation of cluster formation with both laminin and agrin results early on in a quantitatively synergistic response compared with induction solely with laminin or agrin (Fig. 6 A). Furthermore, the rat α7β1 integrin was localized at >60% of the AChR clusters induced by laminin and agrin (Fig. 6 B). As the total numbers of clusters formed in response to agrin and laminin were so enhanced, this extent of coclustering is much greater than expected if agrin induced AChR aggregation independently of the integrin. By 18 h, the α7β1 integrin is detected at essentially all AChR clusters that form in the presence of laminin and agrin (Fig. 4). These results suggest that agrin, laminin, and the α7β1 integrin can participate jointly in the formation of AChR clusters and neuromuscular junctions.


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)

Cluster formation and colocalization of the α7 integrin 5 h after induction of transfected C2C12 myofibers with laminin, agrin,  or laminin and agrin. (A) The number of clusters that form in response to agrin and laminin exceeds that produced by either alone. (B)  Exogenous α7 does not localize well with agrin-induced clusters. In contrast, the synergistic response evoked by agrin and laminin is  characterized by localization of the α7β1 integrin with AChRs, even early in cluster formation. These results suggest that laminin, agrin  and the α7β1 integrin interact in a common or convergent pathway to form AChR clusters and neuromuscular junctions. The mean values scored in triplicate samples, ±SE, are given.
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Related In: Results  -  Collection

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

Figure 6: Cluster formation and colocalization of the α7 integrin 5 h after induction of transfected C2C12 myofibers with laminin, agrin, or laminin and agrin. (A) The number of clusters that form in response to agrin and laminin exceeds that produced by either alone. (B) Exogenous α7 does not localize well with agrin-induced clusters. In contrast, the synergistic response evoked by agrin and laminin is characterized by localization of the α7β1 integrin with AChRs, even early in cluster formation. These results suggest that laminin, agrin and the α7β1 integrin interact in a common or convergent pathway to form AChR clusters and neuromuscular junctions. The mean values scored in triplicate samples, ±SE, are given.
Mentions: It was recently suggested that laminin and agrin initiate clustering by alternative pathways that can be distinguished both temporally and biochemically: agrin induces clustering more rapidly than laminin and requires MuSK (Sugiyama et al., 1997). We therefore examined early on in the induction process whether we could distinguish the association of the α7β1 integrin with agrin- and laminin-induced AChR clusters. Myofibers were treated with agrin, laminin, or with both agrin and laminin, and cluster formation was determined 5 h later. Initiation of cluster formation with both laminin and agrin results early on in a quantitatively synergistic response compared with induction solely with laminin or agrin (Fig. 6 A). Furthermore, the rat α7β1 integrin was localized at >60% of the AChR clusters induced by laminin and agrin (Fig. 6 B). As the total numbers of clusters formed in response to agrin and laminin were so enhanced, this extent of coclustering is much greater than expected if agrin induced AChR aggregation independently of the integrin. By 18 h, the α7β1 integrin is detected at essentially all AChR clusters that form in the presence of laminin and agrin (Fig. 4). These results suggest that agrin, laminin, and the α7β1 integrin can participate jointly in the formation of AChR clusters and neuromuscular junctions.

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