New approaches to identify interactors of MuSK, a receptor tyrosine kinase required for the formation of the nerve-muscle synapse

摘要: Efficient synaptic transmission requires a high local specialization of pre- and postsynaptic cells. At the neuromuscular junction (NMJ), these specializations include aggregates acetylcholine receptors (AChRs). Proteins apparatus implicated in aggregation AChRs utrophin, synapse-specific homolog dystrophin (Ohlendieck et al., 1991; Bewick 1992; Tinsley 1992, 1994), α- s-dystroglycan (Ibraghimov-Beskrovnaya al. Fallon Hall, rapsyn (Frail 1988; Apel 1995), thought to link cytoskeleton. Agrin, heparansulfate proteoglycan that is synthesized by motor neurons deposited into basal lamina, was shown trigger redistribution form (McMahan, 1990; Wallace, 1996; Ruegg Bixby, 1998). It now clear agrin organizes differentiation stimulating MuSK, receptor tyrosine kinase expressed selectively at skeletal muscle (Jennings 1993; Valenzuela 1995; Glass 1996). Agrin MuSK are essential for synapse formation, as mice lacking or fail synapses consequently die birth because failure move breathe (Gautam 1996, DeChiara Nevertheless, mechanisms which activates poorly understood. stimulates rapid phosphorylation myotubes, but, if transiently fibroblast myoblasts, not phosphorylated (Glass et., al These data thus indicate activation depends on least one additional component myotubes but myoblasts. The current hypothesis predicts this component, termed muscle-associated specificity (MASC; 1996), together with an complex. In thesis, we investigated how signal transmitted further downstream leading accumulation synapse. first part, aimed identify proteins associated using membrane bound split-ubiquitin system, method based yeast two-hybrid (YTH) system. contrast original YTH novel allows screen pass it. step, showed bait pray both containing constitutively active reporter genes self dimerization indicating system valuable identifying components intact MuSK. Moreover, also demonstrated soluble used YTH-screen correctly does generate false-positive signals can be screening. After validation experiments, total number 3x107 clones were screened resulting >5’000 putative candidates. However, none them could reconfirmed in dependency tests. several attempts improve per se decrease the number candidates, forced drop project it turned out had many intrinsic problems solved useful time window. In second project, therefore concentrated mapping sites important its MuSK α-dystroglycan binding property. As agrin’s AChR clustering activity is mediated most C-terminal laminin G-like domain region. Moreover, particular splice variant exon 8 amino acids length within the B/z site LG3 potent isoform, whereas variants lacking insert B/z all (Gesemann Guided by crystal structure derived from different variants (Stetefeld al., 2004), analyzed contribution single B/z-8 activate MuSK show activity resides mostly side chains three-peptide motif ‘Asn-Glu-Ile’, highly conserved between species. addition, demonstrate that flanking strongly contribute activity. Finally, we demonstrate affinity α-DG positively affects its activity. Based propose model where plays auxiliary role capturing at the surface efficiently presents molecule complex. In summary, results reported thesis step elucidate detailed mechanism of instructs structures. similar mechanisms are work formation brain, likely important for furthering understanding structures formed altered during development process learning memory.