TY - JOUR
T1 - NGF stimulation of erk phosphorylation is impaired by a point mutation in the transmembrane domain of trkA receptor
AU - Monshipouri, Mariam
AU - Jiang, Hao
AU - Lazarovici, Philip
PY - 2000
Y1 - 2000
N2 - The nerve growth factor (NGF) trkA receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic tyrosine kinase region by a single transmembrane domain (TMD). To explore the role of TMD in the process of receptor activation, we substituted the hydrophobic amino-acid residue valine 432 with the charged amino-acid glutamic acid (designated V432E mutant) by utilizing in vitro site-directed mutagenesis. NIH 3T3 cells lacking endogenous NGF receptors were stably transfected with a pRc/CMV vector carrying either wild- type (trkA) or mutated (V432E) receptors. Stable transfectants were shown, using 125I-NGF binding and Western-blot analysis, to express the trkA recombinant receptors. Scatchard analysis revealed similar affinity for NGF in wild-type and V432E receptors. Although the level of basal trkA receptor tyrosine phosphorylation was higher in the mutant than in the wild-type, NGF stimulation of WT 11 and V432E transfectants resulted in a rapid increase in receptor tyrosine phosphorylation and of its intracellular adaptor protein SHC. In contrast to WT11, V432E mutants showed very low levels of NGF-, and moderate levels of FGF-induced erks phosphorylation, respectively. Collectively, these findings suggest that a single substitution (V432E) in the trkA TMD results in a selective impairment of trkA-mediated erks signaling pathway.
AB - The nerve growth factor (NGF) trkA receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic tyrosine kinase region by a single transmembrane domain (TMD). To explore the role of TMD in the process of receptor activation, we substituted the hydrophobic amino-acid residue valine 432 with the charged amino-acid glutamic acid (designated V432E mutant) by utilizing in vitro site-directed mutagenesis. NIH 3T3 cells lacking endogenous NGF receptors were stably transfected with a pRc/CMV vector carrying either wild- type (trkA) or mutated (V432E) receptors. Stable transfectants were shown, using 125I-NGF binding and Western-blot analysis, to express the trkA recombinant receptors. Scatchard analysis revealed similar affinity for NGF in wild-type and V432E receptors. Although the level of basal trkA receptor tyrosine phosphorylation was higher in the mutant than in the wild-type, NGF stimulation of WT 11 and V432E transfectants resulted in a rapid increase in receptor tyrosine phosphorylation and of its intracellular adaptor protein SHC. In contrast to WT11, V432E mutants showed very low levels of NGF-, and moderate levels of FGF-induced erks phosphorylation, respectively. Collectively, these findings suggest that a single substitution (V432E) in the trkA TMD results in a selective impairment of trkA-mediated erks signaling pathway.
KW - Erk
KW - Impairment
KW - Mutation
KW - NGF
KW - Recombinant receptor
KW - Transmembrane domain
KW - TrkA
KW - Tyrosine kinase
UR - http://www.scopus.com/inward/record.url?scp=0034039955&partnerID=8YFLogxK
U2 - 10.1385/jmn:14:1-2:069
DO - 10.1385/jmn:14:1-2:069
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C2 - 10854038
AN - SCOPUS:0034039955
SN - 0895-8696
VL - 14
SP - 69
EP - 76
JO - Journal of Molecular Neuroscience
JF - Journal of Molecular Neuroscience
IS - 1-2
ER -