Novel functional interactions between Trk kinase and p75 neurotrophin receptor in neuroblastoma cells.
To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET-R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR), and the TrkA transmembrane and intracellular domains. EGF activated the ET-R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation but enhanced the EGF-induced response, leading to differentiation of almost all cells. A mutated NGF, 3T-NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF-induced differentiation required at least nanomolar concentrations of NGF, consistent with the low-affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhanced apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF seems to enhance survival through the phosphatidylinositol-3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET-R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase-3. An anti-p75 antibody blocked the survival effect of NGF. Brain-derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.
Lachyankar
Mahesh B
MB
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Condon
Peter J
PJ
Daou
Marie-Claire
MC
De
Asit K
AK
Levine
John B
JB
Obermeier
Axel
A
Ross
Alonzo H
AH
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D020170:Caspase 1 / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000187:drug effects; D020932:Nerve Growth Factor / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D009447:Neuroblastoma / Q000473:pathology*; D016716:PC12 Cells; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D017510:Protein Folding; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000502:physiology; D050721:Proto-Oncogene Proteins c-cbl; D051381:Rats; D020800:Receptor, Nerve Growth Factor; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000187:drug effects / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D019138:Antibodies, Blocking; D019208:Brain-Derived Neurotrophic Factor; D002352:Carrier Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D004815:Epidermal Growth Factor; D020932:Nerve Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D020917:Receptor, trkA; D020929:Mitogen-Activated Protein Kinase Kinases; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1; C067419:CBL protein, human