Signaling-inactive epidermal growth factor receptor/ligand complexes in intact carcinoma cells by quinazoline tyrosine kinase inhibitors

Several inhibitors of EGF receptor (EGFR) tyrosine kinase activity have bee n developed that compete with ATP at its binding site such as the quinazoli nes PD 153035 and ZD 1839 or the 4,5-dianilino-phthalimides DAPH1 and DAPH2 . When tested on human A431 cells, the quinazolines completely blocked EGF- induced receptor phosphorylation at 100 muM, whereas it was inhibited by DA PH1 and DAPH2 by only 20% at 3 muM. Quinazoline-treated A431 as well as tum or cells expressing less EGFR (A549, MDA MB 231, and T47D) bound 3- to 6-fo ld more I-125-labeled EGF than untreated intact control cells. Scatchard an alysis revealed the disappearance of low- and high-affinity EGFR on A431 ce lls upon PD 153035 treatment. A single receptor class of intermediate ligan d binding affinity emerged and its number corresponded to the sum of the tw o classes. DAPH1 and DAPH2 did not change ligand binding properties of EGFR . PD 153035 exerted the most potent effects on EGF binding to A431 or on in hibiting EGF-stimulated growth of rat MTLn3 cells at low ligand concentrati ons. Cross-linking of EGFR on PD 153035-treated A431 cells indicated the fo rmation of inactive dimers that further increased upon addition of EGF. Che mical cross-linking of I-125-labeled EGF to PD 153035-treated A431 cells re vealed increased binding to monomeric and dimeric EGFR. Thus, the quinazoli nes sequestered EGFR plus the ligand into inactive receptor/ligand complexe s. This novel mode of action of quinazoline tyrosine kinase inhibitors may be the basis for their extraordinary potency especially in conditions when the ligand is present in limiting amounts.