STIMULATION OF NEUROPEPTIDE-Y GENE-EXPRESSION BY BRAIN-DERIVED NEUROTROPHIC FACTOR REQUIRES BOTH THE PHOSPHOLIPASE C-GAMMA AND SHC BINDING-SITES ON ITS RECEPTOR, TRKB

In PC12 cells, it has been previously reported that nerve growth facto r stimulates neuropeptide Y (NPY) gene expression. In the current stud y we examined the signalling pathways involved in this effect by trans iently expressing in PC12 cells the receptor (TrkB) for the related ne urotrophin, brain-derived neurotrophic factor (BDNF). BDNF caused a 3- fold induction of luciferase expression from a transiently co-transfec ted plasmid possessing the firefly luciferase gene under the control o f the NPY promoter. This effect of BDNF was completely blocked by eith er a Y484F mutation in TrkB (which blocks high-affinity Shc binding to TrkB) or by a Y785F substitution [which blocks the binding, phosphory lation and activation of phospholipase Cy (PLC gamma)]. Activation of the NPY promoter by neurotrophin-3 in PC12 cells overexpressing TrkC w as also completely blocked by a naturally occurring kinase insert whic h prevents the high-affinity binding of Shc and PLC gamma. NPY promote r activation by BDNF was blocked by PD98059, suggesting a role for mit ogen-activated protein kinase (MAP kinase). Stimulation of NPY gene ex pression by PMA, but not by BDNF, was blocked by Ro-31-8220, a protein kinase C inhibitor, excluding a role for this serine/threonine protei n kinase in the effect of BDNF. In addition, BDNF did not cause an ele vation in cytosolic Ca2+ concentration. Taken together, our results su ggest that stimulation of the NPY promoter by BDNF requires the simult aneous activation of two distinct pathways; one involves Shc and MAP k inase, and the other appears to be PLC gamma-independent but requires an intact tyrosine-785 on TrkB and so may involve an effector of TrkB signalling that remains to be identified.