Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunofluorescence localization of rat α7  integrin isoforms (green) in mouse C2C12 myofibers  at sites of laminin-induced clusters of acetylcholine  receptors (red) using rat-specific anti-α7 antibody.  The rat α7AX2 and α7BX2 isoforms, but not the  α7AX1 or BX1 isoforms, colocalized with  rhodamine–bungarotoxin at AChR clusters. Endogenous α7 integrin in cells transfected with the pBK  vector and control C2C12 cells does not react with  the 026 antibody. Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132957&req=5

Figure 2: Immunofluorescence localization of rat α7 integrin isoforms (green) in mouse C2C12 myofibers at sites of laminin-induced clusters of acetylcholine receptors (red) using rat-specific anti-α7 antibody. The rat α7AX2 and α7BX2 isoforms, but not the α7AX1 or BX1 isoforms, colocalized with rhodamine–bungarotoxin at AChR clusters. Endogenous α7 integrin in cells transfected with the pBK vector and control C2C12 cells does not react with the 026 antibody. Bar, 15 μm.

Mentions: To study the colocalization of individual α7 integrin isoforms with the laminin-induced AChR clusters, C2C12 mouse myoblasts were transfected with cDNAs encoding rat α7AX1, AX2, BX1, and BX2 under control of the mouse MCK promoter. This promoter is active in differentiated myotubes but not in myoblasts (Jaynes et al., 1986). The rat-specific mouse anti-α7 mAb O26 was used to detect the rat α7 isoforms expressed in mouse myotubes. This antibody also enables more definitive localization and quantitation of the integrin than afforded by conventional antisera. Western blot analysis (not shown) and immunofluorescence (Fig. 2) showed that the transfected cell populations produced equivalent amounts of the appropriate isoforms of rat α7 protein with the exception that α7BX1 was ∼1.5–2-fold higher. The rat integrin was not detected by immunoblot or immunofluorescence in untransfected C2C12 cells or in control cells transfected with the parent plasmid pBKRSV. All the cell populations differentiated at approximately the same time and were morphologically indistinct from the C2C12 control cells. Likewise, all cell populations also formed approximately equivalent numbers of AChR clusters.


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)

Immunofluorescence localization of rat α7  integrin isoforms (green) in mouse C2C12 myofibers  at sites of laminin-induced clusters of acetylcholine  receptors (red) using rat-specific anti-α7 antibody.  The rat α7AX2 and α7BX2 isoforms, but not the  α7AX1 or BX1 isoforms, colocalized with  rhodamine–bungarotoxin at AChR clusters. Endogenous α7 integrin in cells transfected with the pBK  vector and control C2C12 cells does not react with  the 026 antibody. Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Immunofluorescence localization of rat α7 integrin isoforms (green) in mouse C2C12 myofibers at sites of laminin-induced clusters of acetylcholine receptors (red) using rat-specific anti-α7 antibody. The rat α7AX2 and α7BX2 isoforms, but not the α7AX1 or BX1 isoforms, colocalized with rhodamine–bungarotoxin at AChR clusters. Endogenous α7 integrin in cells transfected with the pBK vector and control C2C12 cells does not react with the 026 antibody. Bar, 15 μm.
Mentions: To study the colocalization of individual α7 integrin isoforms with the laminin-induced AChR clusters, C2C12 mouse myoblasts were transfected with cDNAs encoding rat α7AX1, AX2, BX1, and BX2 under control of the mouse MCK promoter. This promoter is active in differentiated myotubes but not in myoblasts (Jaynes et al., 1986). The rat-specific mouse anti-α7 mAb O26 was used to detect the rat α7 isoforms expressed in mouse myotubes. This antibody also enables more definitive localization and quantitation of the integrin than afforded by conventional antisera. Western blot analysis (not shown) and immunofluorescence (Fig. 2) showed that the transfected cell populations produced equivalent amounts of the appropriate isoforms of rat α7 protein with the exception that α7BX1 was ∼1.5–2-fold higher. The rat integrin was not detected by immunoblot or immunofluorescence in untransfected C2C12 cells or in control cells transfected with the parent plasmid pBKRSV. All the cell populations differentiated at approximately the same time and were morphologically indistinct from the C2C12 control cells. Likewise, all cell populations also formed approximately equivalent numbers of AChR clusters.

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