STRUCTURAL DOMAINS OF AGRIN REQUIRED FOR CLUSTERING OF NICOTINIC ACETYLCHOLINE-RECEPTORS

Agrin is an extracellular matrix component which promotes the clusteri ng of nicotinic acetylcholine receptors (nAChRs) and other proteins at the neuromuscular junction. This aggregation process is one of the ea rliest steps in synapse formation. Expression of highly active isoform s of agrin, generated by alternative splicing, is restricted to neuron s in the central nervous system (CNS) including motoneurons. In the ex periments reported here we investigate the regions of agrin necessary for nAChR clustering activity using two different methods. First, we e xpressed truncated soluble forms of the agrin protein in mammalian cel ls and assessed their clustering activity. Second, we generated a pane l of monoclonal antibodies (mAbs) against agrin and mapped their epito pes. Several mAbs block agrin-induced aggregation of nAChRs. One of th e mAbs, Agr86, binds exclusively to the CNS-specific splicing variants and thus identifies an epitope common only to these more active isofo rms. Mapping of the Agr86 epitope suggests that alternative splicing r esults in a distributed conformational change in the agrin protein. Ta ken together our data suggest that four domains in the C-terminal 55 k Da of agrin contribute to its nAChR clustering activity.