K-252B POTENTIATION OF NEUROTROPHIN-3 IS TRKA SPECIFIC IN CELLS LACKING P75(NTR)

K-252b potentiates the neurotrophic effects of neurotrophin-3 (NT-3) i n primary cultures of rat central cholinergic and peripheral sensory n eurons and in a rat pheochromocytoma PC12 cell line. The ligand and re ceptor specificity, and role of the low-affinity neurotrophin receptor (p75(NTR)) in the potentiation response induced by K-252b, are unknow n, To address the issues of ligand and receptor specificity of K-252b potentiation, we have examined neurotrophin-induced DNA synthesis ([H- 3]-thymidine incorporation) in NIH3T3 cells expressing trkA, trkB, or trkC. Neither NT-3 nor K-252b alone could stimulate mitogenic activity in the trkA-overexpressing clone. However, coaddition of K-252b (EC(5 0) of similar to 2 nM) with 10-100 ng/ml NT-3 led to incorporation of [3H]thymidine in trkA expressing cells to a level induced by optimal c oncentrations of nerve growth factor (NGF). The K-252b- and NTS-induce d [H-3]thymidine incorporation correlated with an increase in the tyro sine autophosphorylation of the trkA receptor as well as tyrosine phos phorylation of trk-associated phospholipase C-gamma 1 and SH2-containi ng proteins. K-252b did not potentiate submaximal doses of NGF, or max imal doses of brain-derived neurotrophic factor (BDNF) or neurotrophin -4/5 (NT-4/5) in trkA-expressing cells. Furthermore, K-252b did not po tentiate DNA synthesis by submaximal doses of BDNF, NT-4/5, or NT-3 in trkB- or trkC-expressing NIH3T3 cells, suggesting that the potentiati on profile for K-252b was specific for NT-3 in trk-expressing cells. W e found no expression of p75 NTR in the trk-expressing NIH3T3 cells, T his is the first demonstration that K-252b potentiates a trkA-mediated biological nonneuronal response by NT-3 that occurs independent of p7 5(NTR) and appears to be both ligand and receptor specific.