Functional expression of TrkA receptors in hippocampal neurons.
Nerve growth factor (NGF) initiates its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. The requirements for NGF signaling through the TrkA receptor have been defined extensively from studies in immortalized cells, involving transfection of NIH 3T3, COS, and PC12 cells. In the present study, we tested the effects of extracellular and intracellular mutations of TrkA after DNA-mediated transfection in primary cultures of embryonic day 17 hippocampal neurons. We found that the action of the TrkA receptor on neuronal differentiation depends on specific motifs in the extracellular domain and on tyrosine 490 (Y490), the site for SHC protein binding. In contrast with previous observations in a PC12 background, a mutation in the SHC Y490 binding site in TrkA resulted in a loss of NGF-dependent process formation. These results indicate that tyrosine 490 is necessary for neurite outgrowth in hippocampal neurons. Moreover, a constitutively active form of TrkA did not give enhanced responsiveness in hippocampal neurons, indicating that the behavior of TrkA receptors in primary neuronal cells is distinct from that of other cell types.
Aibel
L
L
Skirball Institute, New York University Medical Center, New York 10016, USA.
Martin-Zanca
D
D
Perez
P
P
Chao
M V
MV
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D009154:Mutation; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051966:Phospholipase C gamma; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020801:Receptor, Ciliary Neurotrophic Factor; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D007527:Isoenzymes; D009414:Nerve Growth Factors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA; D010738:Type C Phospholipases; D051966:Phospholipase C gamma