AGRIN INHIBITS NEURITE OUTGROWTH BUT PROMOTES ATTACHMENT OF EMBRYONICMOTOR AND SENSORY NEURONS

Agrin is a secreted glycoprotein with the ability to cluster cell surf ace molecules, including the nicotinic acetylcholine receptor (AchR) o n muscle cells. Alternate splicing of agrin mRNA results in a family o f agrin proteins which differ in their clustering potency. Neuronal-sp ecific isoforms with the highest clustering activity play a role in cl ustering postsynaptic proteins at the neuromuscular junction. However, the function of agrin isoforms expressed in many nonneuronal tissues, and only weakly active in clustering assays, remains obscure. Monolay er cultures of Chinese hamster ovary (CHO) cells expressing a neuronal (agrin-19) or a nonneuronal (agrin-0) form of agrin were used to assa y the effect of agrin on neurite outgrowth and cell attachment. These results were compared to outgrowth on control CHO cells expressing onl y drug resistance and on regions of CHO-agrin monolayers not expressin g detectable levels of agrin. Neurite extension on confluent monolayer s of agrin-0- or -19-expressing CHO cells was reduced substantially be low that of controls. In one experiment neurite lengths were compared at 2 and 3 days after plating and suggested that neurite outgrowth may be stopped and not simply retarded. Attachment of sensory or motoneur ons was nearly twofold higher to agrin monolayers than to control cell s, showing that the inhibition is not a result of a nonpermissive envi ronment. An agrin construct missing the C-terminal half, removing the major site of variability and clustering activity, was also tested. Th is construct did not reduce outgrowth, suggesting that the C-terminal half of the protein may be important in stopping growth as well as ind ucing clustering. These results expand the role of agrin in synaptogen esis as it may provide a stop signal at the myofiber surface and may a nchor the presynaptic fibers to the eventual motor endplate. (C) 1997 Academic Press