Heterodimerization of the epidermal-growth-factor (EGF) receptor and ErbB2and the affinity of EGF binding are regulated by different mechanisms

When clathrin-dependent endocytosis is inhibited in HeLa cells by overexpre ssion of a K44A (Lys(44) --> Ala) mutant of the GTPase dynamin, high-affini ty binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) is d isrupted [Ringerike, Stang, Johannessen, Sandnes, Levy and Madshus (1998) J . Biol, Chem, 273, 16639-16642]. We now report that the effect of [K44A]dyn amin on EGF binding was counteracted by incubation with the non-specific ki nase inhibitor staurosporine (SSP), implying that a protein kinase is respo nsible for disrupted high-affinity binding of EGF upon overexpression of [K 44A]dynamin, The effect of [K44A]dynamin on EGF binding was not due to alte red phosphorylation of the EGFR, suggesting that the activated kinase is re sponsible for phosphorylation of a substrate other than EGFR. The number of EGFR molecules was increased in cells overexpressing [K44A]dynamin, while the number of proto-oncoprotein ErbB2 molecules was unaltered. EGF-induced receptor dimerization was not influenced by over expression of [K44A]dynami n. ErbB2-EGFR heterodimer formation was found to be ligand-independent, and the number of heterodimers was not altered by overexpression of [K44A]dyna min. Neither SSP nor the phorbol ester PMA, which disrupts high-affinity EG F-EGFR interaction, had any effect on the EGFR homo- or hetero-dimerization , Furthermore, the EGF-induced tyrosine phosphorylation of ErbB2 was not af fected by overexpression of [K44A]dynamin. implying that EGFR-ErbB2 dimers were fully functional. Our results strongly suggest that high-affinity bind ing of EGF and EGFR-ErbB2 heterodimerization are regulated by different mec hanisms.