Article Figures & Data
Figures
- Figure 1.
CD4 chimeras containing the β subunit intracellular loop cocluster with the AChR. A , Schematic showing construction of chimeric proteins, in which the CD4 extracellular and transmembrane domains were fused with the major intracellular (IC) loop of each AChR subunit. B , CD4-subunit loops were expressed transiently in Sol8 myotubes and then immunoprecipitated from cell extracts and immunoblotted with an antibody to the CD4 extracellular domain. All CD4-subunit loop chimeras were robustly expressed. C , Sol8 myotubes transfected with CD4-subunit loop chimeras were treated with agrin for 16 h, and then immunostained for surface CD4 and AChR. Only CD4-β loop coclustered with the AChR in response to agrin treatment (arrows). CD4 and all other CD4-subunit loops remained diffusely distributed on the myotube surface (CD4-γ and δ loops are not shown, but see Table 1). CD4-εCT denotes an additional chimera that includes the IC loop, fourth transmembrane domain and C terminus, which also failed to cluster. Scale bar, 20 μm.
- Figure 2.
A 20 amino acid motif in the β subunit loop is sufficient for clustering. A , CD4-β loop chimeras were generated with either the first (amino acids 333–406) or second halves (407–469) of the loop fused after the CD4 transmembrane domain, and with β333–406 or 407–469 substituted into the corresponding region of ε loop. Agrin induced clustering of CD4-β333–406 and -β333–406/ε (arrows) but not -β407–469 or ε/β407–469 chimeras. CD4-β370–406 also clustered. B , CD4-chimeras were also generated with β loop fragments fused after the CD4 intracellular tail (CD4ct-). Progressive deletions identified a minimal construct (CD4ct-β382–401) that clustered efficiently. Scale bar, 20 μm. C , CD4ct-β382–401 colocalized precisely with the AChR in thin optical sections (Apotome slice ∼0.9 μm). Scale bar, 20 μm. D , Schematic showing β loop structure and CD4-β loop chimeras found to be sufficient for clustering. Y390 denotes a conserved tyrosine phosphorylation site and the thickened line represents the predicted α-helix.
- Figure 3.
Agrin-induced clustering of CD4-β loop chimeras is phosphorylation-dependent. A , CD4-β loop constructs were immunoprecipitated from extracts of control and agrin-treated Sol8 myotubes (C-Ag4,8, 1 nm, ∼1 h) and immunoblotted with a phospho-specific antibody to Y390. Agrin induced phosphorylation of CD4-β, β333–406 and β333–406/ε. Reprobing with mAb124 to the β loop confirmed similar levels of chimeric proteins in each of the immunoprecipitations. B , CD4-β333–406 and CD4-β333–406 Y390F were immunoprecipitated from myotube extracts and immunoblotted with Y390 phospho-specific antibody. Mutation of Y390 (to F) eliminated basal and agrin-induced phosphorylation, without affecting expression levels shown by reprobing with antibody to CD4. C , Agrin-induced clustering of CD4-β loop, β333–406 and β333–406/ε was abolished by Y390F mutation. Scale bar, 20 μm.
- Figure 4.
Overexpression of CD4-β loop chimeras inhibits agrin-induced clustering of the endogenous AChR. A , CD4-β loop chimeras and AChR were sequentially isolated from extracts of transfected Sol8 myotubes using anti-CD4 antibody (CD4 IP) and biotinylated α-bungarotoxin (BuTX) respectively. Levels were then compared by immunoblotting with antibodies to CD4 and the β loop (mAb124). B , Quantification of the mAb124 immunoblots shows that the CD4-β loop constructs were expressed at levels ∼2.5–5 times that of the endogenous AChR, and that mutation of Y390 did not affect expression. Reprobing with antibody to the AChR α subunit also demonstrates that the CD4-β loop chimeras do not directly associate with AChR or assemble with the other receptor subunits. C , Immunostaining for surface CD4 (green) and AChR (red) shows that compared with control (CD4 expressing) myotubes, the number of agrin-induced AChR clusters is reduced in myotubes overexpressing CD4ct-β382–401. In addition, the remaining AChR clusters were sometimes fragmented and less intensely stained (arrows) compared with those in untransfected or CD4 expressing myotubes (arrowheads). Scale bar, 20 μm. D , Quantification of the number of agrin-induced AChR clusters in myotubes overexpressing CD4 or CD4-β loop chimeras. Compared with CD4-expressing control myotubes, the number of AChR clusters was significantly reduced (∼25%) in myotubes expressing CD4-β370–406 and CD4ct-β382–401 (p < 0.01, Student's t test, n = 8). In contrast, no inhibition occurred in myotubes expressing β370–406 and β382–401 with Y390F mutations.
- Figure 5.
Rapsyn is preferentially associated with phosphorylated AChR. A , Surface AChR were isolated from control and agrin-treated Sol8 myotubes using biotinylated α-bungarotoxin (BuTX) and immunoblotted with βY390 phospho-specific antibody, anti-α subunit antibody (mAb210) and anti-rapsyn antibody (B6766). Agrin treatment for 1 h (C-Ag4,8, 200 pm) increased both the amount of rapsyn that coimmunoprecipitated with the AChR and β subunit phosphorylation. B , Phosphorylated AChR was immunoprecipitated with phospho-βY390 antibody, and then residual surface AChR was isolated using biotinylated α-BuTX. Immunoblotting with anti-PY antibodies shows that most AChR containing phosphorylated β subunit was immunoprecipitated with phospho-specific βY390 antibody, and reprobing with mAb210 shows that ∼1/2 of the total surface receptor is phosphorylated. Immunoblotting for rapsyn indicates that more rapsyn is associated with phosphorylated than nonphosphorylated AChR.
- Figure 6.
Rapsyn binds β loop 382–401 in a phosphorylation-dependent manner. A , CD4ct-β382–401 chimeras were immunoprecipitated from extracts of control or agrin plus pervanadate-treated myotubes (500 pm agrin, 0.2 mm pervanadate; 1 h) and immunoblotted with anti-rapsyn antibody (B5668). Rapsyn associated with wild-type CD4ct-β382–401 only after agrin plus pervanadate treatment that induced robust phosphorylation of Y390. No rapsyn associated with Y390F CD4ct-β382–401 that was expressed at similar levels but lacked phosphorylation (n = 4). Sequential isolation and immunoblotting of the AChR showed that agrin plus pervanadate induced significantly greater β subunit phosphorylation than agrin alone. B , GST fusion proteins immobilized on membranes were probed with 5 μg/ml GST-rapsyn and bound protein detected by immunoblotting with anti-rapsyn antibody (B5668). Rapsyn bound specifically to GSTct-β382–401 Y390 and Y390F but not to GST or GSTct controls, and binding was eliminated by incubating the GST-rapsyn probe together with 25 μg/ml soluble GSTct-β382–401. Coomassie staining shows that all fusion proteins were present at similar levels (n = 5).
- Figure 7.
Phosphorylation of β loop increases the stoichiometry of rapsyn binding to AChR. A , B , Surface AChR was pulled down from wild type and AChR-β3F/3F myotubes using biotinylated α-BuTX and the isolates immunoblotted with anti-α subunit (mAb210) and anti-rapsyn antibody (B6766). Agrin increased the amount of rapsyn associated with wild type AChR (∼1.8-fold, p < 0.01, n = 6, ANOVA with Tukey post-test) but not AChR-β3F/3F (ns = not significant). In addition, less rapsyn was associated with AChR-β3F/3F compared with wild type AChR in both control and agrin-treated conditions (p < 0.05 and 0.001, respectively, ANOVA with Tukey post-test). C , D , Rapsyn was immunoprecipitated from wild type and AChR-β3F/3F myotubes and the isolates immunoblotted with anti-α subunit (mAb210) and anti-rapsyn antibody (B6766). Agrin did not affect the amount of wild type or β3F/3F AChR associated with rapsyn (n = 6, differences not significant, ANOVA with Tukey post-test). Thus, agrin-induced β phosphorylation increases the ratio of rapsyn to AChR, but not AChR to rapsyn, suggesting that it primarily increases stoichiometry of rapsyn binding to each AChR.
- Figure 8.
Working model for how agrin-induced phosphorylation of AChR β subunit regulates rapsyn association and receptor localization. A , Alignment showing that the β subunit motif sufficient for clustering is highly conserved between species. B , Our data together with previous findings suggest a model in which AChR localization involves both constitutive and regulated interactions with rapsyn. In uninnervated muscle, rapsyn (R) is constitutively associated with the AChR, interacting with the helical domain (H) of one or more subunit loops. On innervation, agrin-induced phosphorylation of the AChR β subunit (βP) induces binding of additional rapsyn to the β 382–401 motif. This increase in stoichiometry may increase the efficiency of AChR clustering and also anchor and stabilize the receptor via interactions with dystroglycan (DG) or cytoskeletal proteins. Both forms of rapsyn binding contribute to localizing the AChR at high density in the postsynaptic membrane.
Tables
- Table 1.
Quantification of agrin-induced clustering of CD4-subunit loops and β loop fragments
CD4-chimera No. of experiments No. of myotubes counted % myotubes with clusters CD4 5 80 1 CD4− α loop 5 88 0 β loop 6 94 73 γ loop 3 57 0 δ loop 5 63 3 ε loop 5 75 0 β333–406 6 117 74 β407–469 5 84 2 β333–406/ε 6 111 77 β370–406 3 60 45 β loop Y390F 6 100 1 β333–406 Y390F 3 57 2 β333–406/ε Y390F 3 53 0 β370–406 Y390F 3 60 3 CD4ct− β382–401 3 60 88 β382–401 Y390F 3 60 15 β385–396 3 60 5 δ385–404 3 60 22 -
CD4-chimera-positive myotubes were identified in random fields and scored for the presence or absence of CD4 clusters that colocalized with AChR clusters. Agrin induced robust clustering of CD4-β loop chimeras encompassing amino acids 382–401, with 45–88% of the expressing myotubes having clusters. Mutation of Y390 abolished clustering of these constructs, and all other CD4-loops showed background or weak colocalization with AChR clusters.
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