SELECTIVE REGULATION OF AGRIN MESSENGER-RNA INDUCTION AND ALTERNATIVESPLICING IN PC12 CELLS BY RAS-DEPENDENT ACTIONS OF NERVE GROWTH-FACTOR

The extracellular matrix protein agrin plays an important role in the formation and maintenance of the neuromuscular junction, However, regu lation of agrin gene expression and pre-mRNA splicing, important in de termining the biological actions of agrin, is not well understood, To begin to identify mechanisms controlling agrin expression, quantitativ e polymerase chain reaction techniques were used to analyze the effect of growth factors on the expression of agrin mRNA isoforms in rat phe ochromocytoma (PC12) cells. Agrin transcripts in untreated cells lacke d inserts in the Y and Z sites (agrin(y0z0)), encoding agrin isoforms with low acetylcholine receptor aggregating activity and a primarily n on-neuronal tissue distribution, Transcripts encoding isoforms with hi gh aggregating activity and neuronal tissue distribution (agrin(y4z8) agrin(y4z11), and agrin(y4z19)) were not detected, Treatment of PC12 c ells with nerve growth factor (NGF) caused a significant increase in t otal agrin mRNA, In contrast, exposure to epidermal growth factor had no effect, Analysis of alternative splicing of agrin mRNA revealed tha t NGF elicited a specific increase in agrin(y4) and agrin(z8) mRNAs th at did not occur in the presence of epidermal growth factor, insulin, dexamethasone, or retinoic acid. Analysis of PC12 sublines stably over expressing a dominant inhibitory form of p21 Ras indicated that NGF in duced changes in levels of agrin mRNA and alternative splicing require d Ras activity, The results show that NGF can influence important aspe cts of neuronal differentiation by regulating alternative splicing, Fu rthermore, these data provide insight into the mechanisms governing ag rin gene expression and suggest that neurotrophic factors may play a r ole in regulating agrin expression in vivo.