Pathogenic activation of receptor tyrosine kinases in mammalian membranes.
The mechanism of receptor tyrosine kinase (RTK) over-activation due to mutations in their transmembrane (TM) domain is not well understood, and different mechansims have been proposed to contribute to pathogenesis. Here, we address the effect of two such pathogenic mutations (V664E in Neu and A391E in fibroblast growth factor receptor 3 (FGFR3)) on receptor activation in mammalian cells. We develop a quantitative description of receptor activation in terms of free energies of activation, and generate mathematical predictions of active fractions as a function of receptor expression. We test the mathematical predictions by comparing them to Western blot measurements of active fractions of Neu and chimeric Neu_FGFR3 receptors in CHO cells. We show that the predictions describe the experimental data, thus yielding a quantitative measure of receptor over-activation due to the two mutations studied. In CHO cells, the V664E mutation increases the Neu activation propensity by about -1.1 kcal/mol, while the increase due to the A391E mutation is about -0.7 kcal/mol. The two values are similar, and likely represent Glu-mediated stabilization of the active dimeric state. Thus, an increase of the order of approximately -1 kcal/mol may be sufficient to transform normal signaling processes into pathogenic processes. The results of this study increase our knowledge of the mechanism behind RTK-mediated pathologies, and highlight the potential utility of inhibitors that target the dimerization process. They also suggest the cellular response to stimuli can be understood and predicted based on quantitative knowledge of interaction strengths between proteins involved in signaling.
He
Lijuan
L
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
Hristova
Kalina
K
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D008322:Mammals / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000378:metabolism; D013816:Thermodynamics; D014162:Transfection
D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D020794:Receptor Protein-Tyrosine Kinases; D018719:Erb-b2 Receptor Tyrosine Kinases; D051498:Receptor, Fibroblast Growth Factor, Type 3