Defining the structural basis for acetylcholine receptor clustering activity of neural agrin.

摘要

Multiple forms of agrin that differ in binding properties and bioactivity are synthesized by nerve and muscle. Motoneuron-derived agrin containing an eight amino acid insert (z8) introduced by alternative splicing of Z exon is critical for induction of synaptic differentiation at the neuromuscular junction. Muscle isoforms of agrin lacking the insert exhibit little activity and are dispensable during synaptogenesis in vivo. In this study it is demonstrated that a synthetic z8 peptide neither induced AChR clustering on myotube surface nor competed with neural agrin. Alanine scanning mutagenesis of z8 sequence in agrin demonstrated the receptor-clustering activity is correlated with the ability of protein to interact with calcium. Calcium induced a striking change in the conformation of neural agrin, but not the muscle isoform. Substitution of the 4th asparagine residue (N4) in z8 abolished the structural change, decreased the binding affinity, and diminished AChR-clustering activity of neural agrin. Mutation of two aspartic acids in the calcium-binding site (D1820 and D1889) also significantly reduced the protein activity. The z8 insert forms a loop situated in proximity to the aspartates, and residue N4 maybe able to interact with calcium. In conclusion, alternative splicing at Z exon confers AChR-clustering activity by promoting calcium-dependent folding of neural agrin.