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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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The α7AX2 and α7BX2 integrins exhibit a high level of  colocalization with laminin-induced AChR clusters in transfected  C2C12 myofibers. H36 anti-α7 antibody added at the time of induction inhibits this colocalization. A low level of colocalization  is seen with AChR clusters and the α7AX1 and α7AX2 isoforms.  The mean values scored in triplicate samples, ±SE, are given.
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Figure 3: The α7AX2 and α7BX2 integrins exhibit a high level of colocalization with laminin-induced AChR clusters in transfected C2C12 myofibers. H36 anti-α7 antibody added at the time of induction inhibits this colocalization. A low level of colocalization is seen with AChR clusters and the α7AX1 and α7AX2 isoforms. The mean values scored in triplicate samples, ±SE, are given.

Mentions: Quantitation of colocalization confirms our visual observations. The extent of colocalization of α7 isoforms and AChR clusters promoted by laminin was analyzed in three separate experiments by scoring AChR clusters and determining the number of clusters with α7 integrin. Clusters with <50% coincident α7 or β1 integrin and AChR signals were not scored as colocalized. By this stringent criterion, >80% of the AChR clusters were colocalized with α7AX2 and α7BX2 integrins upon induction with 60 nM laminin (Fig. 3). This increase was not observed in α7AX1 or α7BX1 transfected cell lines, again indicating an essential role for the α7X2 extracellular domain in laminin-induced AChR clustering. When antibody specific for the rat α7 integrin was added to cultures at the same time cluster formation was initiated with laminin, the anti-α7 antibody inhibited colocalization of the rat integrin with AChRs. Furthermore, this inhibition was restricted to colocalization of the α7AX2 and α7BX2 isoforms.


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)

The α7AX2 and α7BX2 integrins exhibit a high level of  colocalization with laminin-induced AChR clusters in transfected  C2C12 myofibers. H36 anti-α7 antibody added at the time of induction inhibits this colocalization. A low level of colocalization  is seen with AChR clusters and the α7AX1 and α7AX2 isoforms.  The mean values scored in triplicate samples, ±SE, are given.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: The α7AX2 and α7BX2 integrins exhibit a high level of colocalization with laminin-induced AChR clusters in transfected C2C12 myofibers. H36 anti-α7 antibody added at the time of induction inhibits this colocalization. A low level of colocalization is seen with AChR clusters and the α7AX1 and α7AX2 isoforms. The mean values scored in triplicate samples, ±SE, are given.
Mentions: Quantitation of colocalization confirms our visual observations. The extent of colocalization of α7 isoforms and AChR clusters promoted by laminin was analyzed in three separate experiments by scoring AChR clusters and determining the number of clusters with α7 integrin. Clusters with <50% coincident α7 or β1 integrin and AChR signals were not scored as colocalized. By this stringent criterion, >80% of the AChR clusters were colocalized with α7AX2 and α7BX2 integrins upon induction with 60 nM laminin (Fig. 3). This increase was not observed in α7AX1 or α7BX1 transfected cell lines, again indicating an essential role for the α7X2 extracellular domain in laminin-induced AChR clustering. When antibody specific for the rat α7 integrin was added to cultures at the same time cluster formation was initiated with laminin, the anti-α7 antibody inhibited colocalization of the rat integrin with AChRs. Furthermore, this inhibition was restricted to colocalization of the α7AX2 and α7BX2 isoforms.

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