Lrp4 is a receptor for Agrin and forms a complex with MuSK

Abstract

Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin.

Figure 1. Lrp4 is required for Agrin to stimulate MuSK phosphorylation

(A) Agrin stimulates MuSK tyrosine phosphorylation in wild-type but not in lrp4 mutant myotubes. (A, B) Restoring Lrp4 expression in lrp4 mutant myotubes rescues Agrin-stimulated MuSK phosphorylation as well as AChR clustering. (C) Expression of LDLR fails to rescue MuSK phosphorylation. (D) Flag-Lrp4 is not tyrosine phosphorylated by Agrin stimulation, indicating that Lrp4 is not itself a substrate for MuSK. (E) A Lrp4/LDLR chimera, in which the LDLR cytoplasmic domain was exchanged for the Lrp4 cytoplasmic domain, rescues Agrin-stimulated MuSK phosphorylation and AChR clustering.

Figure 2. Lrp4 is a receptor for Agrin

(A) BaF3 cells, expressing Flag-Lrp4-mCherry, myc-MuSK-GFP, Flag-LDLR, CCR7-GFP, or parental BaF3 cells, were perfused through a chamber containing a glass coverslip on which Agrin B8, BSA or laminin (LN) had been adsorbed. (B) Bright field (BF), fluorescence (mCherry) and interference reflection microscopy (IRM) were used to determine the number of cells that bound to each substrate. The darkest area of the IRM image marks the close apposition of BaF3 cells to Agrin or antibodies. By all criteria, BaF3 cells expressing Lrp4, or Lrp4 and MuSK, bound selectively to Agrin. (C) Binding of Lrp4 to Agrin is specific, since BaF3 cells expressing myc-MuSK-GFP or Flag-LDLR, as well as parental BaF3 cells and cells expressing CCR7-GFP, fail to bind Agrin. Antibodies to epitope tags, introduced into the ectodomain of Lrp4 (Flag), MuSK (myc) and LDLR (Flag), were also adsorbed to the glass coverslip and served as positive and negative controls. (D) Quantitation of the number (mean ± s.e.m.) of Flag-Lrp4-mCherry, myc-MuSK-GFP, Flag-LDLR and parental BaF3 cells that bound to Agrin B8, BSA, LN, anti-Flag and anti-myc, as determined by BF and IRM optics.

Figure 3. Lrp4 binds selectively and with high affinity to neural isoforms of Agrin

(A) The graph shows the gates for distinguishing cells expressing: Lrp4-mCherry, MuSK-GFP, Lrp4-mCherry plus MuSK-GFP, as well as parental BaF3 cells, as measured by flow cytometry. (B) The histograms depict binding of Cy5-Agrin B8 to the gated populations of cells shown in (A) at 100 nM Agrin. Cy5-Agrin B8 bound preferentially to cells expressing Lrp4-mCherry or Lrp4-mCherry plus MuSK-GFP and weakly to cells expressing MuSK-GFP alone. (C) Lrp4-expressing cells bound Agrin B8 with ~100-fold higher affinity (~6 nM) than Agrin B0. Binding between Agrin B8 and Lrp4 is strengthened ~20-fold in the presence of Ca⁺⁺. The inset shows binding at the lower concentrations of Agrin. The 95 kd and 50 kd forms of Agrin B8 bound similarly to cells expressing Lrp4 or Lrp4 and MuSK (data not presented). Agrin binding to parental cells was minimal (B) and was subtracted from all values. (D) Co-expression of MuSK-GFP potentiates binding of Cy5-Agrin B8 to cells expressing Lrp4-mCherry; addition of the soluble ectodomain from MuSK (2 µM) also enhances binding of Cy5-Agrin B8 to Lrp4-mCherry expressing cells.

Figure 4. Lrp4 self-associates and forms a complex with MuSK

(A) BaF3 cells, as well as BaF3 cells expressing myc-MuSK-GFP or CCR7-GFP, grow in suspension as single cells. Flag-Lrp4-mCherry-expressing cells, however, form aggregates, indicating that Lrp4 binds Lrp4. Although myc-MuSK-GFP-expressing cells grow as solitary cells when cultured alone, they co-aggregate with Flag-Lrp4-mCherry expressing cells, indicating that MuSK binds Lrp4. (B) Binding between Lrp4 and MuSK is specific, since myc-MuSK-GFP-expressing cells fail to aggregate with themselves, and BaF3 cells expressing CCR7-GFP fail to co-aggregate with Flag-Lrp4-mCherry-expressing cells. (C) Lrp4 and MuSK interact in cis, as they co-immunoprecipitate when expressed together in the same cell.

Figure 5. Agrin stimulates MuSK phosphorylation in non-muscle cells expressing Lrp4

(A) Lrp4, like MuSK, is expressed in C2 and Sol8 myotubes but poorly, if at all in myoblasts. (B) Agrin stimulates phosphorylation of MuSK-GFP in BaF3 cells expressing Lrp4-mCherry and MuSK-GFP but not in cells expressing MuSK-GFP alone. (C) Agrin stimulates MuSK phosphorylation of in BaF3 cells expressing Lrp4, MuSK-GFP and Dok-7 but not in cells expressing MuSK-GFP and Dok-7 without Lrp4. MuSK was immunoprecipitated from BaF3 cells with antibodies to MuSK, and Western blots were probed with antibodies to phosphotyrosine or GFP.

Figure 6

Cartoon illustrating interactions between neural Agrin, Lrp4 and MuSK. Lrp4 self-associates and interacts with MuSK in the absence of Agrin (left). The arrangement of Lrp4 and MuSK within this complex is not known, and the complex may be oligomeric rather than dimeric, as depicted. Moreover, the domains responsible for the Lrp4-Agrin and Lrp4-MuSK interactions are not known. Neural Agrin binds to the preformed complex and triggers a reorganization or reorientation of MuSK, promoting trans-phosphorylation and kinase activation (right). Once phosphorylated, MuSK activates a signaling pathway that leads to synaptic differentiation, including clustering of AChRs. Domain coloring for Lrp4: white, LDLa; orange, EGF-like; blue, b-propeller. Domain coloring for MuSK: purple, immunoglobulin-like; yellow, Frizzled-like cysteine-rich; red or green, tyrosine kinase (TK).