Conjugation of LG domains of agrins and perlecan to polymerizing laminin-2 promotes acetylcholine receptor clustering.
Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and betaAChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development.
Smirnov
Sergei P
SP
Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Barzaghi
Patrizia
P
McKee
Karen K
KK
Ruegg
Markus A
MA
Yurchenco
Peter D
PD
D018171:Agrin / Q000737:chemistry*; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D016000:Cluster Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005821:Genetic Techniques; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry*; D007797:Laminin / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D032342:Muscle Cells / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D009469:Neuromuscular Junction / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D011108:Polymers / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011950:Receptors, Cholinergic / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013569:Synapses / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D018171:Agrin; D018076:DNA, Complementary; D019812:Heparan Sulfate Proteoglycans; D007797:Laminin; D008562:Membrane Glycoproteins; D011108:Polymers; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; C029967:nidogen; C071927:perlecan; D014443:Tyrosine