K‐252b Potentiation of Neurotrophin‐3 Is trkA Specific in Cells Lacking p75^NTR

Abstract

K-252b potentiatesthe neurotrophic effects of neurotrophin-3 (NT-3) in primary cultures of rat central cholinergic and peripheral sensory neurons and in a rat pheochromocytoma P012 cell line. The ligand and receptor specificity, and role of the low-affinity neurotrophin receptor (p75NTR) in the potentiation response induced by K-252b, are unknown. To address the issues of ligand and receptor specificity of K-252b potentiation, we have examined neurotrophin-induced DNA synthesis ([ 3H]-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 (E0 50 of~-2 nM) with 10-1 00 ng/ml NT-3 led to incorporation of[ 3H]thymidine in trkA expressing cells to a level induced by optimal concentrations of nerve growth factor (NGF). The K252b-and NT-3-induced [3H]thymidineincorporation correlated with an increase in the tyrosine autophosphorylation of the trkA receptor as well as tyrosine phosphorylation of trk-associated phospholipase C-yl and SH2-containing proteins. K-252b did not potentiate submaximal doses of NGF, or maximal doses of brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-41 5) in trkA-expressing cells. Furthermore, K-252b did not potentiate DNA synthesis by submaximal doses of BDNF, NT-4/5, or NT-3 in trkB-or trkC-expressing NIH3T3 cells, suggesting that the potentiation profile for K-252b was specific for NT-3 in trkA-expressing cells. We found no expression of p75 NTR in the trk-expressing NIH3T3 cells. This is the first demonstration that K-252b potentiates a trkA-mediated biological nonneuronal response by NT-3 that occurs independent of p75 NTR and appears to be both ligand and receptor specific. Key Words: K-252b-Potentiation-trk-p75 low-affinity nerve growth factor receptor.

Inhibition of Glial Cell Line‐Derived Neurotrophic Factor Induced Intracellular Activity by K‐252b on Dopaminergic Neurons

“…K-252b, structurally and actively related to K-252a, inhibits NGF action on choline acetyltransferase (ChAT) activity in cholinergic cells (Knüsel and Hefti, 1991) and BDNF action on DA uptake in ventral mesencephalon cultures (KnUsel et al, 1992a). It, however, has also been shown to selectively potentiate cellular actions of NT-3 (Knusel et al, 1992a;Maroney et al, 1997). In addition, K-252b prevents phosphatidylinositol breakdown and phosphorylation of phospholipase Cy 1 (PLC-yl) mediated by BDNF and NT-3 (Widmer et al, 1993a, b).…”

CEP-1347 promotes survival of NGF responsive neurones in primary DRG explants

“…These factors include a range of growth factors and neurotrophin mimetic compounds. These compounds include the alkaloid K252a [12,20], K252b, which has no biological activity when added alone but potentiates the activity of neurotrophin-3 [21,22], and CEP-1347, the K252a derivative [7,8], which inhibits MLK and promotes survival of neurones both in vitro and in vivo.…”

Quantitative, High-Throughput Cell-Based Assays for Inhibitors of trkA Receptor

“…NIH3T3 cells that were transfected with the pMex-neo vector containing the full-length rat cDNA of trkA (11) were provided by Dr. Anna C. Maroney (Cephalon, Inc., West Chester, PA). The cell line transfection and characterization of the trk-transfected cells have been previously described (12). NIH3T3 cells transfected with trkA (NIH3T3-trkA) or pMex-neo vector (NIH3T3-vector) were maintained in growth medium (DMEM supplemented with 2 mM glutamine, 10% bovine calf serum, 55 g/ml geneticin, and 50 U/ml penicillin-streptomycin) at 37°C in 10% CO 2 , 90% air atmosphere.…”