Crk/Crk-L-mediated Rapgef1 recruitment couples MuSK signaling to AChR anchoring at the neuromuscular junction.

The neuromuscular junction (NMJ) is established through reciprocal signaling between motoneurons and skeletal muscle, a process critically dependent on the receptor tyrosine kinase MuSK. Although MuSK activation by Agrin, Lrp4, and Dok-7 is well characterized, the temporal organization of downstream signaling remains unclear. Here, we define the MuSK proximity proteome using TurboID-based proximity labeling combined with mass spectrometry. We identified 74 MuSK-associated proteins, including known substrates and previously unreported interactors. Among early signaling components, the adapter Crk-L emerged as a central hub linking MuSK to small GTPase pathways. We further identified Rapgef1 as a Crk-L-associated effector required for Agrin-dependent acetylcholine receptor (AChR) phosphorylation and clustering. Rapgef1 loss in adult muscle led to NMJ fragmentation. We propose that these defects result from reduced cytoskeletal anchorage of AChRs due to compromised Rapsyn/AChR interaction. These findings uncover Rapgef1 as a key regulator of MuSK-mediated signaling and highlight dynamic mechanisms governing NMJ formation and maintenance.