ABSTRACT
This study examined the effects of hypomorphic p75 neurotrophin receptor (p75NTR) expression and high levels of nerve growth factor (NGF) on trkA phosphorylation and downstream activation of p44/42 mitogen-activated protein kinase (MAPK). Post-ganglionic sympathetic neurons from postnatal day 1 p75NTR exon III null mutant (p75(-/-)) and 129/SvJ mice were cultured in the presence of 50 ng/mL NGF and analysed by Western blotting. Levels of phosphorylated trkA are increased in p75(-/-) neurons compared with 129/SvJ neurons, and these higher levels are maintained with continuous exposure to NGF. MAPK is also phosphorylated to a greater extent in p75(-/-) neurons than in 129/SvJ neurons, both within 10 min of exposure to NGF, and with continuous NGF treatment for 5 days. These data provide new insight into the mechanism underlying enhanced neurite outgrowth in p75(-/-) neurons, demonstrating that trkA and MAPK signalling in sympathetic neurons are increased when p75NTR function is disrupted.
Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neurons/metabolism , Receptor, trkA , Receptors, Nerve Growth Factor/deficiency , Superior Cervical Ganglion/cytology , Animals , Animals, Newborn , Blotting, Western/methods , Cell Count/methods , Cells, Cultured , Mice , Mice, Inbred Strains , Mice, Knockout , Nerve Growth Factor/pharmacology , Phosphorylation , Precipitin Tests/methods , Rats , Receptor, Nerve Growth Factor , Receptors, Nerve Growth Factor/genetics , Receptors, Nerve Growth Factor/metabolism , Time FactorsABSTRACT
The neurotrophin nerve growth factor (NGF) binds to two receptor types: the tyrosine kinase receptor TrkA and the common neurotrophin receptor p75(NTR). Although many of the biological effects of NGF (such as neuronal growth and survival) are associated with TrkA activation, p75(NTR) also contributes to these activities by enhancing the action of TrkA when receptors are coexpressed. The NGF antagonist PD90780 [7-(benzolylamino)-4,9-dihydro-4-methyl-9-oxo-pyrazolo[5,1-b]quinazoline-2-carboxylic acid] interacts with NGF, preventing its binding to p75(NTR). In this study, the actions of this compound are further explored, and it is found that PD90780 is not able to inhibit the binding of either brain-derived neurotrophic factor or neurotrophin-3 to p75(NTR), consistent with the direct interactions of the antagonist with NGF. In addition, we demonstrate that the ability of PD90780 to inhibit NGF-p75(NTR) interactions is lower when receptors are coexpressed, compared with when p75(NTR) is the only neurotrophin receptor expressed. These results suggest that the interaction between NGF and the p75(NTR) receptor is altered when TrkA is coexpressed. This alteration can be exploited in the development of antagonists that will selectively inhibit the pro-apoptotic actions of p75(NTR) when expressed in the absence of TrkA, although having less effect on the pro-survival effects of p75(NTR) mediated by enhanced TrkA activation.
