(A)–(C) Interaction of LRP4 and neuronal agrin in solution. Beads were conjugated with Flag-nAgrin, which were subsequently incubated with condition media of HEK293 cells expressing LRP4N-Myc (A), MuSKect-Myc (B), LRP6N-Myc (C), or empty vector (control). Bound proteins were isolated by bead precipitation, resolved by SDS-PAGE and visualized by immunoblotting with anti-Myc antibody. Flag-nAgrin interacted with LRP4N-Myc (A), but not MuSKect-Myc (B) or LRP6N-Myc (C).
(D) Interaction of Wnt-1 and LRP6N. Beads were conjugated with Wnt-1-HA, which were subsequently incubated with LRP6N-Myc. Bound LRP6N-Myc was revealed by immunoblotting.
(E) No interaction between Wnt-1 and LRP4N. Beads were conjugated with Wnt-1-HA, which were subsequently incubated with LRP4N-Myc. Bound LRP4N-Myc was revealed by immunoblotting.

(A) Neuronal, but not muscle, agrin bound to intact C2C12 myoblasts transfected with LRP4. C2C12 myoblasts were transfected by empty vector (control), LRP4 and/or Flag-MuSK. 36 hr after transfection, myoblasts were incubated with AP alone, mAgrin-AP or nAgrin-AP for 90 min at room temperature. Endogenous AP was inactivated by heating and bound AP was visualized in cells by staining with BCIP/NBT.
(B) Quantification of data in A. Data shown were mean ± SEM. n = 6; *, P < 0.05 in comparison with mAgrin-AP of the same group or nAgrin-AP in the control group.
(C, D) nAgrin-AP bound to HEK293 cells expressing LRP4, but not those expressing LRP5. HEK293 cells were transfected without (control) or with LRP4-Myc (C) or LRP5-Myc (D). 36 hr after transfection, transfected cells were incubated with nAgrin-AP or mAgrin-AP. In some experiments, control cells were incubated with nAgrin-AP. After heat inactivation of endogenous AP, lysates were assayed for transfected AP using pNPP as substrate. Lysates were also subjected to immunoblotting to reveal the expression of different amounts of LRP4-Myc (C) and LRP5-Myc (D). Data shown were mean ± SEM. n = 6.
(E, F) LRP4 expression enabled MuSK and Abl activation by agrin in HEK293 cells. Cells were transfected with LRP4 and/or Flag-MuSK (E) or Flag-Abl (F). 36 hr after transfection, cells were treated without or with neuronal agrin for 1 hr and were then lyzed. In E, lysates were incubated with anti-Flag antibody, and resulting immunocomplex was analyzed with anti-phosphotyrosine antibody 4G10. In F, active Abl was revealed by immunoblotting with specific phospho-Abl antibody. Lysates were also blotted for Flag and/or Myc, LRP4, or β-actin to indicate equal amounts of proteins.
(G) Quantitative analysis of data in E and F. MuSK and Abl phosphorylation was quantified by using the ImageJ software. Data shown were mean ± SEM. n = 3; *, P < 0.05 in comparison with control.

(A) Increased LRP4-MuSK interaction in the presence of neuronal agrin. Flag-MuSKect immobilized on beads were incubated with condition media of cells expressing the extracellular domains of LRP4 (LRP4N-Myc) or the empty vector (control) in the presence or absence of neuronal agrin. Precipitated LRP4 was analyzed by immunoblot with anti-Myc antibody. Reaction mixtures were also blotted directly for Flag and Myc to demonstrate equal amounts of proteins.
(B) Quantitative analysis of LRP4N-Myc and Flag-MuSK. Data shown were mean ± SEM, n = 3; *, p < 0.05 in comparison with the no-agrin group.
(C) Dose-dependent interaction between LRP4 and MuSK. Purified LRP4-Myc was coated on Maxi-Sorp Immuno Plates, which were incubated with MuSK-AP. Bound AP was measured with pNPP as substrate. Data shown were mean ± SEM. n = 4.
(D) Scatchard plot of data in C. Y axis represents the ratio of bound to free MuSK-AP whereas X axis represents the concentration of bound MuSK-AP.
(E) No interaction of LRP6 and MuSK extracellular domains. Experiments were done as in A except condition medium of cells expressing the extracellular domain of LRP6 was used.
(F) Co-immunoprecipitation of LRP4 and MuSK. HEK293 cells were transfected with LRP4 and/or Flag-MuSK. Lysates were incubated with anti-Flag antibody, and resulting immunocomplex was analyzed for LRP4 and Flag. Lysates were also probed to indicate equal amounts of indicated proteins.
(G) Interaction of LRP4 with MuSK in mouse muscles. Mouse muscles of indicated ages were homogenized, and homogenates were incubated with rabbit anti-LRP4 antibody or rabbit normal IgG. Precipitates were probed for MuSK and LRP4. Homogenates were also probed directly for MuSK, LRP4, and β-actin (bottom panels).

(A) Temporal expression pattern of LRP4 during muscle differentiation. C2C12 myoblasts were switched to the differentiation medium. Muscle cells were collected at indicated times and lyzed. Lysates (30 µg of protein) were resolved by SDS-PAGE and visualized by immunoblotting using indicated antibodies.
(B) Colocalization of LRP4 with R-BTX in muscle sections. Diaphragm sections were incubated with polyclonal antibodies against LRP4 or MuSK, which h was visualized by Alexa Fluor 488-conjugated anti-rabbit antibody. R-BTX was included in the reaction to label postsynaptic AChRs. Arrows indicate co-localization of LRP4 or MuSK with AChRs.
(C) Enrichment of LRP4 in synaptic regions of muscles. Synaptic (S) and non-synaptic (NS) regions of hemi-diaphragms were isolated and homogenized. Homogenates (30 µg of protein) were analyzed for LRP4 or AChR (as control) using specific antibodies. Samples were also probed for β-actin to indicate equal loading.

(A) Schematic diagrams of AP constructs. Neuronal or muscle agrin was fused to AP in pAPtag-5. The fusion proteins contain a signal peptide (SS) in the N-terminus, and two additional tags (Myc and His) in the C-terminus. Neuronal agrin contains 4- and 8-amino acid residue inserts at the Y and Z sites, respectively.
(B) Functional characterization of agrin-AP recombinant proteins. C2C12 myotubes were stimulated with AP alone, mAgrin-AP or nAgrin-AP for 18 hr. AChR clusters were assayed as described in Experimental Procedures. Data shown were mean ± SEM. n = 4; *, P < 0.05 in comparison with AP or mAgrin-AP.
(C) Differential binding activities of mAgrin-AP and nAgrin-AP to myoblasts and myotubes. C2C12 myoblasts and myotubes were incubated AP alone, mAgrin-AP or nAgrin-AP for 90 min at room temperature. Endogenous AP was inactivated by heating and bound AP was assayed by staining with BCIP/NBT. Data shown were mean ± SEM. n = 6; *, P < 0.05.
(D) Direct interaction between LRP4 and neuronal agrin. LRP4-Myc was purified and coated on Maxi-Sorp Immuno Plates, which were incubated with nAgrin-AP or mAgrin-AP. AP activity was measured with pNPP as substrate. Control, condition medium of HEK293 cells transfected with the empty pAPtag-5. Data shown were mean ± SEM. n = 3; *, P < 0.05 in comparison with AP or mAgrin-AP.
(E) Dose-dependent interaction between LRP4 and neuronal Agrin. Purified LRP4-Myc was coated on Maxi-Sorp Immuno Plates, which were incubated with nAgrin-AP or mAgrin-AP. AP activity was measured with pNPP as substrate. Data shown were mean ± SEM. n = 4; *, P < 0.05.
(F) Scatchard plot of data in E. Y axis represents the ratio of bound to free nAgrin-AP whereas X axis represents the concentration of bound nAgrin-AP.

(A) Characterization of LRP4-miRNA constructs. HEK293 cells were transfected with LRP4 and LRP4-miLRP4 constructs or control miRNA that encoded scrambled sequence. Cell lysates were analyzed for LRP4 expression by immunoblotting with anti-LRP4 antibody. β-Actin was used as loading control. miLRN4-1062 was most potent in inhibiting LRP4 expression.
(B) Repression of LRP4 expression reduced neuronal agrin binding to myotube surface. C2C12 myotubes were transfected with control (scramble) miRNA or miLRP4-1062. Cells were incubated with AP, mAgrin-AP or nAgrin-AP, which was visualized in cell as described in Figure 3A.
(C) Quantitative analysis of data in B. Data shown were mean ± SEM. n = 6; *, p < 0.05 in comparison nAgrin-AP with control.
(D) MuSK activation by neuronal agrin was diminished in C2C12 myotubes transfected with miLRP4-1062. C2C12 myotubes were transfected with control miRNA or miLRP4-1062. 36 hr later, myotubes were treated without or with agrin for 1 hr and cells were then lyzed. MuSK was isolated by immunoprecipitation and blotted with the anti-phosphotyrosine antibody 4G10. Lysates were also blotted for MuSK, LRP4, GFP (encoded by miRNA constructs), and β-actin to indicate equal amounts of proteins.
(E) Quantitative analysis of data in D by ImageJ software (mean ± SEM, n = 3; *, P < 0.05 in comparison with control).
(F) Neuronal agrin-induced clustering of AChRs was inhibited in C2C12 myotubes transfected with miLRP4-1062. C2C12 myotubes were transfected by control miRNA, miLRP4-1062, miMuSK-1161, or miLRP5-1490. AChR clusters were induced by neuronal agrin and quantified as described in Experimental Procedures (mean ± SEM, n = 5; *, p < 0.05 in comparison with control). miMuSK-1161 and miLRP5-1490 were able to suppress expression of respective proteins in transfected cells (data not shown).

(A) Agrin stimulated the interaction between endogenous LRP4 and MuSK. C2C12 myotubes were stimulated without or with neuronal agrin. Lysates were subjected to immunoprecipitation with rabbit anti-LRP4 antibody (top panels) or rabbit normal IgG (middle panels). Resulting precipitates were probed for MuSK or LRP4. Lysates were also probed with antibodies against LRP4, MuSK, or β-actin to demonstrate equal amounts (bottom panels).
(B) Quantitative analysis of data in A by using the ImageJ software (mean ± SEM, n = 3; *, P < 0.05 in comparison with the no-agrin group).
(C) Agrin stimulated tyrosine phosphorylation of LRP4 in muscle cells. C2C12 myotubes were treated without or with agrin for 1 hr. Lysates were subjected to immunoprecipitation with antibodies against LRP4 and MuSK, respectively. Resulting precipitates were probed with anti-phospho-tyrosine antibody 4G10, or antibodies against LRP4 and MuSK, respectively, to indicate equal amounts of precipitated proteins.
(D) Quantitative analysis of data in C. Data shown were mean ± SEM, n = 3; *, p < 0.05 in comparison with no-nAgrin.
(E) A working model. In the absence of neuronal agrin, LRP4 could interact with MuSK and this interaction is increased by agrin stimulation. Such interaction is necessary for MuSK activation and downstream signaling that leads to AChR clustering. P, phosphorylation.