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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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Colocalization of the rat α7 integrin is largely restricted  to laminin-induced AChR clusters. The exogenously derived integrin is localized at virtually all laminin-induced clusters, but at  only 10–20% agrin-induced clusters. In contrast, laminin and  agrin together induce ∼30% more clusters than do either separately, and the α7 integrin is associated with essentially all clusters. As in Figs. 1–3, localization of the integrin and AChRs was  determined 18 h after induction. The mean values scored in triplicate samples, ±SE, are given.
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Figure 4: Colocalization of the rat α7 integrin is largely restricted to laminin-induced AChR clusters. The exogenously derived integrin is localized at virtually all laminin-induced clusters, but at only 10–20% agrin-induced clusters. In contrast, laminin and agrin together induce ∼30% more clusters than do either separately, and the α7 integrin is associated with essentially all clusters. As in Figs. 1–3, localization of the integrin and AChRs was determined 18 h after induction. The mean values scored in triplicate samples, ±SE, are given.

Mentions: To examine whether the integrin is also present at agrin-induced AChR clusters, colocalization of α7 isoforms and AChR clusters promoted by agrin, laminin, and by a combination of laminin and agrin, was analyzed. Induction with 0.5 nM agrin resulted in relatively low levels of receptor colocalization (8–19%) in myotubes expressing any of the α7 chain isoforms (Fig. 4). In contrast, more than 90% of the AChR clusters were colocalized with α7AX2 and α7BX2 integrins upon induction with 60 nM laminin. The numbers of clusters that formed upon incubation with both 0.5 nM agrin and 60 nM laminin were 30–74% greater than those induced separately by each protein. Anti-α7 antibody will also inhibit this enhancement (see below). Moreover, upon induction with both laminin and agrin virtually all clusters colocalize with the α7AX2 and α7BX2 integrin isoforms. Taken together, these results suggest that laminin, agrin, and the α7β1 integrin interact in a common or convergent pathway. In this pathway, either inducer can independently promote AChR clustering, whereas together, they promote a more vigorous response, with the α7β1 integrin at all 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)

Colocalization of the rat α7 integrin is largely restricted  to laminin-induced AChR clusters. The exogenously derived integrin is localized at virtually all laminin-induced clusters, but at  only 10–20% agrin-induced clusters. In contrast, laminin and  agrin together induce ∼30% more clusters than do either separately, and the α7 integrin is associated with essentially all clusters. As in Figs. 1–3, localization of the integrin and AChRs was  determined 18 h after induction. 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 4: Colocalization of the rat α7 integrin is largely restricted to laminin-induced AChR clusters. The exogenously derived integrin is localized at virtually all laminin-induced clusters, but at only 10–20% agrin-induced clusters. In contrast, laminin and agrin together induce ∼30% more clusters than do either separately, and the α7 integrin is associated with essentially all clusters. As in Figs. 1–3, localization of the integrin and AChRs was determined 18 h after induction. The mean values scored in triplicate samples, ±SE, are given.
Mentions: To examine whether the integrin is also present at agrin-induced AChR clusters, colocalization of α7 isoforms and AChR clusters promoted by agrin, laminin, and by a combination of laminin and agrin, was analyzed. Induction with 0.5 nM agrin resulted in relatively low levels of receptor colocalization (8–19%) in myotubes expressing any of the α7 chain isoforms (Fig. 4). In contrast, more than 90% of the AChR clusters were colocalized with α7AX2 and α7BX2 integrins upon induction with 60 nM laminin. The numbers of clusters that formed upon incubation with both 0.5 nM agrin and 60 nM laminin were 30–74% greater than those induced separately by each protein. Anti-α7 antibody will also inhibit this enhancement (see below). Moreover, upon induction with both laminin and agrin virtually all clusters colocalize with the α7AX2 and α7BX2 integrin isoforms. Taken together, these results suggest that laminin, agrin, and the α7β1 integrin interact in a common or convergent pathway. In this pathway, either inducer can independently promote AChR clustering, whereas together, they promote a more vigorous response, with the α7β1 integrin at all 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