A double cysteine trkA mutant exhibiting reduced NGF binding and delayed Erk signaling.
The NGF receptor trkA is a tyrosine kinase receptor comprising an extracellular domain with a ligand-binding site, a transmembrane-spanning domain (TMD), and an intracellular domain composed of a juxtamembrane region (JMR), a tyrosine kinase domain, and a short carboxy-terminal tail. Nerve growth factor (NGF) binds and activates this receptor, leading to phosphorylation of signaling substrates involved in neuronal proliferation, differentiation, and survival. Human trkA contains one cysteine residue in the TMD (C423) and another, separated by 12 residues, in the JMR (C436). We hypothesized that the removal of one or both of the cysteines would affect NGF-induced signaling of the trkA receptor. Here we show that NGF induces rapid receptor autophosphorylation in a wild-type, trkA-expressing clone (WT11), in a single cysteine trkA mutants (C423T or C436A), but lower autophosphorylation activity in a double-cysteine trkA mutant (C423T/C436A). WT11 and SM cells had similar binding affinity, but that of DM cells was lower, according to the NGF radioreceptor assay. NGF-induced Erk phosphorylation was rapid in WT11 and C423T cells, but delayed in C436A and C423T/C436A cells. NGF induced [3H]thymidine incorporation into WT11 and SM cells, but had no effect on DM cells. However, basic fibroblast growth factor (bFGF) induced rapid phosphorylation of Erk1/2, and [3H]thymidine incorporation in NIH3T3, WT11, single mutant (SM), and double mutant (DM) cells, suggesting that the impaired NGF-induced Erk phosphorylation and thymidine incorporation observed in DM cells are due to the double-cysteine mutations in the trkA receptor. Cumulatively, our findings support a model in which Cys436 of the trkA is responsible for the rapid transfer of the transmembrane occupancy signal to the SHC adaptor protein for activation of the Ras-Erk pathway and DNA synthesis.
Jiang
H
H
William T. Gossett Neurology Laboratories, Henry Ford Health Sciences Center, Detroit, MI 48202, USA.
Movsesyan
V
V
Liu
X W
XW
Katagiri
Y
Y
Monshipoyri
M
M
Lazarovici
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018929:Cell Culture Techniques; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020932:Nerve Growth Factor / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D020917:Receptor, trkA / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013936:Thymidine / Q000378:metabolism; D014162:Transfection
D016222:Fibroblast Growth Factor 2; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D003545:Cysteine; D013936:Thymidine