Journal: The Journal of biological chemistry
Article Title: The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.
doi: 10.1016/j.jbc.2023.104962
Figure Lengend Snippet: Figure 7. Domains of LRP4 interacting with ColQ. A, schematic representation of ectoLRP4-AP and its deletion mutants. B, plate-binding assays. Same concentrations of ectoLRP4-AP and of the indicated deletion mutants were added to ColQ-coated or CT wells. Bound proteins were quantified by measuring AP activity. Results are expressed as the mean ± SEM percentage of the control value set as 100% (ectoLRP4-AP bound to CT wells). n ≥6; **p < 0.01; ****p < 0.0001, using two-way ANOVA followed by Tukey’s multiple comparison post hoc test. For interaction factor: F = 18.07, p < 0.0001; for CT versus ColQ: F = 162, p < 0.0001; for the comparison of the different ectoLRP4 mutants: F = 21.01, p < 0.0001. The N-terminal region of LRP4 plays a crucial role in ColQ–LRP4 interaction as its deletion (ectoLRP4Δ1-AP) compromised binding to ColQ. Conversely, the N-terminal region alone (ectoLRP4Δ234-AP) bound to ColQ at the same level as ectoLRP4-AP. C, ColQ-coated wells were incubated with 25 nM of ectoLRP4-AP in the presence or not of 500 nM purified re- combinant neural agrin. Results are the mean ± SEM percentage of ectoLRP4-AP bound to ColQ wells in the absence of agrin (set as 100%; n = 9, ***p < 0.001, using one-sample t test). The binding of ecto-LRP4 to ColQ was reduced by more than 50% in the presence of agrin. D, pull-down assay where ColQ- coated beads were incubated with 500 nM of His-tagged recombinant neural agrin. Agrin signals were analyzed by Western immunoblot using antibodies against His-tag. No agrin was coprecipitated with ColQ. AP, alkaline phosphatase; ColQ, collagen Q; LRP4, low-density lipoprotein receptor–related protein 2; ns, not significant.
Article Snippet: After 5 days of differentiation, WT and ColQ-deficient myotubes were stimulated or not with 5 nM of recombinant rat neural agrin (R&D Systems) for 16 h. Muscle cells were fixed in 4% paraformaldehyde and incubated with Alexa Fluor 594– conjugated α-bungarotoxin (1:1000 dilution) for 1 h. Images were collected using a microscope (Olympus BX61) equipped with a Fast 1394 Digital CCD FireWire camera (model Retiga 2000R; Qimaging) and a 40× oil immersion objective (numerical aperture: 1.0; Olympus).
Techniques: Binding Assay, Activity Assay, Control, Comparison, Incubation, Pull Down Assay, Recombinant, Western Blot
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