Muscarinic receptor activation stimulates B-50/GAP43 phosphorylation in isolated nerve growth cones

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Abstract
A characteristic feature of neurite formation is high expression of the phosphoprotein B-50/GAP43. Previous studies with growth cone membranes have indicated that this neuron-specific protein kinase C substrate may be involved in transmembrane signal transduction at the growth cone. We monitored the degree of phosphorylation of B-50 by quantitative B-50 immunoprecipitation from intact nerve growth cones, isolated from 5-day-old rat brain and prelabeled with 32P-orthophosphate. B-50 phosphorylation in nerve growth cones is stimulated by 4 beta-phorbol 12,13-dibutyrate (PDB) and 1,2-dioctanoylglycerol (DOG) in a concentration-dependent manner, but not by 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD). These results confirm that B-50 is a substrate of PKC in intact growth cones. Depolarization induced by 30 mM K+ produces a transient increase in B-50 phosphorylation, which is maximal after 15 sec and declines to basal level within 5 min. This rise in B-50 phosphorylation can be partially blocked by atropine (10(-3)-10(-4) M), suggesting the involvement of muscarinic receptors. Indeed, the cholinergic receptor agonist carbachol enhances B-50 phosphorylation in a concentration-dependent manner (50% at 10(-3) M). Since the effect of carbachol (10(-3) M) can be blocked by atropine (10(-7) M), we conclude that this increase in B-50 phosphorylation is mediated through activation of the muscarinic receptors on the growth cones. The carbachol-induced stimulation is further increased by concurrent K+-depolarization. The effects of carbachol and depolarization are additive. To our knowledge, this is the first report showing receptor-mediated effects on the PKC substrate B-50 in growth cones. Our data support the hypothesis that phosphorylation of B-50 by PKC is involved in signal transduction in nerve growth cones.

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