When clathrin-dependent endocytosis is inhibited in HeLa cells by overexpression of a K44A (Lys44 → Ala) mutant of the GTPase dynamin, high-affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) is disrupted [Ringerike, Stang, Johannessen, Sandnes, Levy and Madshus (1998) J. Biol. Chem. 273, 16639–16642]. We now report that the effect of [K44A]dynamin on EGF binding was counteracted by incubation with the non-specific kinase inhibitor staurosporine (SSP), implying that a protein kinase is responsible for disrupted high-affinity binding of EGF upon overexpression of [K44A]dynamin. The effect of [K44A]dynamin on EGF binding was not due to altered phosphorylation of the EGFR, suggesting that the activated kinase is responsible 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 overexpression of [K44A]dynamin. ErbB2–EGFR heterodimer formation was found to be ligand-independent, and the number of heterodimers was not altered by overexpression of [K44A]dynamin. Neither SSP nor the phorbol ester PMA, which disrupts high-affinity EGF–EGFR interaction, had any effect on the EGFR homo- or hetero-dimerization. Furthermore, the EGF-induced tyrosine phosphorylation of ErbB2 was not affected by overexpression of [K44A]dynamin, implying that EGFR–ErbB2 dimers were fully functional. Our results strongly suggest that high-affinity binding of EGF and EGFR–ErbB2 heterodimerization are regulated by different mechanisms.
Abbreviations used: EGF, epidermal growth factor; EGFR, EGF receptor; SSP, staurosporine; PKC, protein kinase C; PKA, cAMP-dependent protein kinase A; FBS, fetal-bovine serum; TBS, Tris-buffered saline [10mM Tris/HCl (pH7.6)/137mM NaCl]; ECL® (Amersham), enhanced chemiluminescence; ECF, enhanced chemifluorescence; BS3, bis(sulphosuccinimidyl) suberate; Cl3Ac, trichloroacetic acid; PTA, phosphotungstic acid; SAB, sodium acetate buffer containing 0.5M NaCl; DMEM, Dulbecco's modified Eagle's medium; MEM, minimal essential medium; PI 3-kinase, phosphoinositol 3-kinase; K44A, Lys44 → Ala; AP, alkaline phosphatase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase.