Spectroscopic studies of the epidermal growth factor and epidermal growth factor receptor system: Native fluorescence and spin-labeling approaches.

摘要

Spectroscopic studies of the epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) system were undertaken using spin-labeled and fluorescently-labeled proteins. Human recombinant EGF was spin-labeled at the N-terminus or the single methionine residue. Spin probes, which were modeled upon the potent and specific anilinoquinazoline class of tyrosine kinase inhibitors, were synthesized as spin probes for EGFR and shown to inhibit EGFR tyrosine kinase activity. Spin-labeled EGFs or spin-labeled inhibitors were added to purified EGFR in detergent solution. In the spin-labeled EGF experiments, the lack of change in the lineshapes of ESR spectra was interpreted as a lack of spin-labeled EGF binding to EGFR under the conditions used, although binding assays performed using A431 cells showed spin-labeled EGFs would bind EGFR under a different set of conditions. In the experiments using spin-labeled inhibitors, the ESR signal obtained, which was invariant in the presence or absence of EGFR protein, was attributed to spin-labeled inhibitor present in the detergent micelle.;In the fluorescence studies, quinazoline and pyridopyrimidine inhibitors were found to possess a native blue or green fluorescence, which is highly solvatochromic and reminiscent of the fluorescence of anilinonaphthalene sulfonate (ANS) dyes. Virtually nonfluorescent in water and other protic solvents, the inhibitors fluoresce in an organic, nonpolar solvent environment or when bound to hydrophobic proteins including EGFR. Models of the photophysical processes present in these systems, including multiple fluorescent excited states, are presented and compared against the fluorescence behavior of ANS. Living A431 human epidermoid carcinoma cells expressing high levels of the EGFR were exposed to low concentrations of the inhibitors and the time-dependent cellular distribution of inhibited EGFR protein was visualized by fluorescence microscopy. These results show that intrinsic fluorescence of the quinazoline and pyridopyrimidine inhibitors can provide a general means for the real-time study of protein-protein interactions and trafficking of EGFR without difficult labeling procedures, chemical modification of the protein, or the generation of mutants, and enables the direct observation in living cells of such biologically and pharmacologically relevant phenomena as uptake and partitioning of inhibitors into subcellular compartments, specific binding to target tyrosine kinases, and inhibitor metabolism.