Agrin binds to the N-terminal region of Lrp4 protein and stimulates association between Lrp4 and the first immunoglobulin-like domain in muscle-specific kinase (MuSK)

Abstract

Neuromuscular synapse formation depends upon coordinated interactions between motor neurons and muscle fibers, leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. Synapse formation begins as motor axons approach muscles that are prepatterned in the prospective synaptic region in a manner that depends upon Lrp4, a member of the LDL receptor family, and muscle-specific kinase (MuSK), a receptor tyrosine kinase. Motor axons supply Agrin, which binds Lrp4 and stimulates further MuSK phosphorylation, stabilizing nascent synapses. How Agrin binds Lrp4 and stimulates MuSK kinase activity is poorly understood. Here, we demonstrate that Agrin binds to the N-terminal region of Lrp4, including a subset of the LDLa repeats and the first of four β-propeller domains, which promotes association between Lrp4 and MuSK and stimulates MuSK kinase activity. In addition, we show that Agrin stimulates the formation of a functional complex between Lrp4 and MuSK on the surface of myotubes in the absence of the transmembrane and intracellular domains of Lrp4. Further, we demonstrate that the first Ig-like domain in MuSK, which shares homology with the NGF-binding region in Tropomyosin Receptor Kinase (TrKA), is required for MuSK to bind Lrp4. These findings suggest that Lrp4 is a cis-acting ligand for MuSK, whereas Agrin functions as an allosteric and paracrine regulator to promote association between Lrp4 and MuSK.

FIGURE 1.

The N-terminal region of Lrp4 is necessary and sufficient to bind Agrin. A, we used a solid-phase binding assay to measure binding of AP-ecto-Lrp4 to wells coated with BSA, non-neural Agrin B0, or neural Agrin B8. An approximately 50-kDa fragment, composed of LDLa domains 6–8, the first two EGF-like domains, and the first of four β-propeller domains, is necessary and sufficient to bind Agrin. Neither the LDLa domains together with the two EGF-like domains nor the four β-propeller domains together with the two EGF-like domains are sufficient to bind neural Agrin. The mean ± S.E. (n ≥ 5) are indicated. B, β-propeller domains 2, 3, or 4 cannot substitute for β-propeller domain 1. Only β-propeller 1, together with the LDLa and EGF-like domains, binds neural Agrin. The mean ± S.E. (n = 2 or 3) are indicated.

FIGURE 2.

Agrin stimulates association between Lrp4 and MuSK. A, we used a solid-phase binding assay to measure binding of AP-ecto-Lrp4 to Fc or MuSK-Fc (M-Fc) in the absence of Agrin or presence of non-neural Agrin B0 or neural Agrin B8. In the absence of Agrin, AP-ecto-Lrp4 binds modestly better (4-fold) to MuSK-Fc than Fc alone (p < 0.01). Agrin B8 selectively stimulated binding between full-length AP-ecto-Lrp4 and MuSK. The mean ± S.E. (n = 4) are indicated. B, neural Agrin stimulated coaggregation between Lrp4-mCherry- and MuSK-GFP-expressing cells. Agrin treatment led to the near complete aggregation of MuSK-GFP cells with Lrp4-mCherry cells and a 5.5-fold increase in the size of the Lrp4-mCherry/MuSK-GFP coaggregates. The mean ± S.E. (n = 4) are indicated.

FIGURE 3.

Binding of MuSK to Lrp4 requires multiple LDLa repeats and β-propeller domains in Lrp4. We used a solid-phase binding assay to measure binding of AP-ecto-Lrp4 and Fc or MuSK-Fc (M) in the presence of neural Agrin B8. Deletion of β-propeller domain 4 in Lrp4 increased binding between Lrp4 and MuSK, suggesting a role for β-propeller domain 4 in restraining association between Lrp4 and MuSK. β-propeller domain 3 is essential for binding between Lrp4 and MuSK. LDLa repeats 4 and 5 contribute to binding between Lrp4 and MuSK. Binding between ecto-Lrp4 and MuSK was assigned a value of 100%, and all other values are expressed relative to ecto-Lrp4. The mean ± S.E. (n ≥ 3) are indicated.

FIGURE 4.

The solvent-exposed surface of the first Ig-like domain in MuSK binds Lrp4. A and B, cells expressing wild-type MuSK-GFP or MuSK L83R-GFP coaggregate with Lrp4-mCherry cells, whereas cells expressing MuSK I96A-GFP cells aggregate poorly with Lrp4-mCherry cells. MuSK-expressing cells fail to aggregate in the absence of Lrp4-expressing cells (3). B, cells expressing Lrp4 and wild-type MuSK form coaggregates of similar size. In contrast, cells expressing MuSK I96A coaggregate poorly with Lrp4-expressing cells. The mean ± S.E. (n = 4) are indicated. C, we used a solid-phase binding assay to measure binding between AP-ecto-Lrp4 and Fc, Fc-MuSK, or Fc-MuSK I96A in the presence of neural Agrin B8. Mutation of Ile-96 in MuSK severely impairs binding to AP-ecto-Lrp4. The mean ± S.E. (n = 3) are indicated.

FIGURE 5.

The extracellular region of Lrp4 is sufficient to stimulate MuSK phosphorylation in myotubes. Addition of the soluble, extracellular region of Lrp4 to lrp4 mutant myotubes restores Agrin-stimulated MuSK phosphorylation. Deletion of the fourth β-propeller domain in Lrp4, which increased binding between Agrin and Lrp4 and enhanced Agrin-dependent association between Lrp4 and MuSK, reduced Agrin-stimulated MuSK phosphorylation by 2-fold (p = 0.055). Additional deletion of the third β-propeller domain in Lrp4, which abolishes binding between Lrp4 and MuSK, prevents Agrin-stimulated MuSK phosphorylation. IP, immunoprecipitation.

FIGURE 6.

Domains in Lrp4 required for binding to Agrin and MuSK. LDLa repeats 6–8, the first β-propeller domain, and the two intervening EGF-like domains in Lrp4 are sufficient to bind neural Agrin. LDLa repeats 4–8, β-propeller domains 1–3, and the two intervening EGF-like domains in Lrp4 are sufficient to bind MuSK.