Uyen Dao_2023_J.Biol.Chem__104962

ColQ is a non-fibrillar collagen that plays a crucial role at the vertebrate neuromuscular junction (NMJ) by anchoring acetylcholinesterase (AChE) to the synapse. ColQ also functions in signaling, as it regulates acetylcholine receptor clustering and synaptic gene expression, in a manner dependent on Muscle-Specific Kinase (MuSK), a key protein in NMJ formation and maintenance. MuSK forms a complex with Low-density lipoprotein receptor-related protein 4 (LRP4), its co-receptor for the proteoglycan agrin at the NMJ. Previous studies suggested that ColQ also interacts with MuSK. However, the molecular mechanisms underlying ColQ functions and ColQ-MuSK interaction have not been fully elucidated. Here, we investigated whether ColQ binds directly to MuSK and/or LRP4 and whether it modulates agrin-mediated MuSK/LRP4 activation. Using co-immunoprecipitation, pull-down, plate binding assays and surface plasmon resonance, we show that ColQ binds directly to LRP4 but not to MuSK, and that ColQ interacts indirectly with MuSK through LRP4. In addition, we show the N-terminal region of LRP4, which contains the agrin binding sites, is also crucial for ColQ binding to LRP4. Moreover, ColQ-LRP4 interaction was reduced in the presence of agrin, suggesting that agrin and ColQ compete for binding to LRP4. Strikingly, we reveal ColQ has two opposing effects on agrin-induced MuSK/LRP4 signaling: it constitutively reduces MuSK phosphorylation levels in agrin-stimulated myotubes, but at the same time increases MuSK accumulation at the muscle cell surface. Together, our results identify LRP4 as a major receptor of ColQ and provide new insights into mechanisms of ColQ signaling and AChE anchoring at the NMJ.