NGF stimulation of erk phosphorylation is impaired by a point mutation in the transmembrane domain of trkA receptor.
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 WT 11, 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.
Monshipouri
M
M
Patent and Trade Mark Office, US Department of Commerce, Washington, DC 20231, USA.
Jiang
H
H
Lazarovici
P
P
D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D018698:Glutamic Acid; D006801:Humans; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020932:Nerve Growth Factor / Q000494:pharmacology*; D010766:Phosphorylation; D017354:Point Mutation; D020917:Receptor, trkA / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014633:Valine
D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D018698:Glutamic Acid; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D020928:Mitogen-Activated Protein Kinases; D014633:Valine