Heterodimerization of the epidermal-growth-factor (EGF) receptor and ErbB2 and the affinity of EGF binding are regulated by different mechanisms.

When clathrin-dependent endocytosis is inhibited in HeLa cells by overexpression of a K44A (Lys(44)-->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.

Epidermal growth factor receptor efficiently activates mitogen-activated protein kinase in HeLa cells and Hep2 cells conditionally defective in clathrin-dependent endocytosis.

Epidermal growth factor (EGF)-induced signaling was investigated in cells conditionally defective in clathrin-dependent endocytosis by overexpression of K44A dynamin in HeLa cells and potassium depletion in Hep2 cells. Overexpression of mutant dynamin disrupts high-affinity EGF-EGF receptor (EGFR) interaction (T. Ringerike, E. Stang, L. E. Johannessen, D. Sandnes, F. O. Levy, and I. H. Madshus, 1998, J. Biol. Chem. 273, 16639-16642). However, the EGFR substrates Shc and c-Cbl were as efficiently tyrosine phosphorylated in endocytosis-deficient HeLa cells exhibiting only low-affinity EGFRs as in HeLa cells with intact endocytosis and with both high- and low-affinity EGFRs. Both Raf and mitogen-activated protein kinase (MAPK) were activated to the same extent and with the same kinetics. HeLa cells distributed equally in the cell cycle regardless of EGFR internalization. Upon potassium depletion of Hep2 cells, EGF-induced EGFR endocytosis was inhibited. However, the EGFR and MAPK were efficiently activated by EGF in both the absence and the presence of clathrin-dependent endocytosis. The EGFR was weakly tyrosine phosphorylated by potassium depletion even in the absence of EGF, and this activation resulted in detectable activation of MAPK. Our results demonstrate that internalization of EGFR by clathrin-dependent endocytosis is not required for activation of MAPK.

Polyubiquitination of the epidermal growth factor receptor occurs at the plasma membrane upon ligand-induced activation.

We have previously shown that, although overexpression of mutant dynamin inhibits clathrin-dependent endocytosis and disrupts high affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR), it does not inhibit ligand-induced translocation of the EGFR into clathrin-coated pits. In the present study, we demonstrate that, upon ligand binding and incubation at 37 degrees C, the EGFR was polyubiquitinated regardless of overexpression of mutant dynamin. In cells not overexpressing mutant dynamin, the EGFR was rapidly internalized and deubiquitinated. In cells being endocytosis-deficient, due to overexpression of mutant dynamin, however, the EGFR was upon prolonged chase first found in deeply invaginated coated pits, and then eventually moved out of the coated pits and back onto the smooth plasma membrane. Polyubiquitination occurred equally efficiently in cells with or without intact clathrin-dependent endocytosis, while the kinetics of ubiquitination and deubiquitination was somewhat different. We further found that the EGF-induced ubiquitination of Eps15 occurred both in the absence and presence of endocytosis with the same kinetics as polyubiquitination of the EGFR, but that the EGF-induced monoubiquitination of Eps15 was somewhat reduced upon overexpression of mutant dynamin. Our data show that EGF-induced polyubiquitination of the EGFR occurs at the plasma membrane.

Epidermal growth factor increases the level of the cyclin-dependent kinase (CDK) inhibitor p21/CIP1 (CDK-interacting protein 1) in A431 cells by increasing the half-lives of the p21/CIP1 transcript and the p21/CIP1 protein.

DNA synthesis was inhibited in A431 cells by epidermal growth factor (EGF) in a p21/CIP1-dependent manner [where CIP1 is cyclin-dependent kinase (CDK)-interacting protein 1]. When 1 or 10 nM EGF was added, the level of p21/CIP1 was increased to the same extent, and the protein level peaked after approx. 5 h of incubation. The increase in p21/CIP1 mRNA upon addition of EGF was rapid, and was enhanced in the presence of cycloheximide. The half-life of p21/CIP1 mRNA in EGF-treated A431 cells was increased approx. 2-fold; this is in contrast with the case in MCF-7 cells with normal p53, in which the half-life of p21/CIP1 mRNA was not increased upon addition of EGF. This increased stability accounts for most of the increase in mRNA levels observed in A431 cells during short incubation periods. Additionally, upon prolonged incubation of A431 cells with EGF, the half-life of the protein was also increased compared with that in untreated cells and in cells treated with EGF for short time periods. Nuclear run-on assays demonstrated only marginal stimulation of transcription by 10 or 1 nM EGF, or by 10 ng/ml tumour necrosis factor alpha. Our results indicate that the most important mechanisms by which EGF increases p21/CIP1 protein levels in A431 cells are post-transcriptional and post-translational stabilization.

Endocytosed epidermal growth factor (EGF) receptors contribute to the EGF-mediated growth arrest in A431 cells by inducing a sustained increase in p21/CIP1.

We investigated the ability of endocytosed activated epidermal growth factor receptors (EGFR) to induce expression of the cyclin-interacting protein p21/CIP1 in A431 cells. Transforming growth factor alpha (TGFalpha) and EGF both induced tyrosine phosphorylation, induction of p21/CIP1, and thereby inhibition of DNA synthesis. TGFalpha is released from the EGFR when the TGFalpha-EGFR complex encounters low pH upon endocytosis. Consistently, we found more rapid dephosphorylation of the EGFR and less induction of p21/CIP1 by TGFalpha than by EGF. This difference was abolished upon neutralizing endosomal pH by the carboxylic ionophore monensin or the proton ATPase inhibitor bafilomycin A1. When surface-bound TGFalpha was removed by acid stripping and endosomal pH was neutralized with bafilomycin A1, TGFalpha stimulated EGFR tyrosine phosphorylation, induced p21/CIP1, and inhibited DNA synthesis. This strongly suggests that p21/CIP1 can be induced by endocytosed, activated EGFR and that endocytosed EGFR can affect cell growth.

High-affinity binding of epidermal growth factor (EGF) to EGF receptor is disrupted by overexpression of mutant dynamin (K44A).

Activation of the epidermal growth factor receptor (EGFR) kinase was analyzed in cells conditionally defective for clathrin-dependent endocytosis by overexpression of mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on ice was strongly reduced in cells overexpressing mutant dynamin, and consistently, binding analyses showed that high-affinity EGFRs were lost. In the absence of mutant dynamin the cells displayed both high- and low-affinity EGFR. At 4 degreesC EGF-EGFR localized mainly outside coated pits regardless of expression of mutant dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37 degreesC. Thus, expression of mutant dynamin disrupts high-affinity binding of EGF, but not ligand-induced recruitment of EGFR to clathrin-coated pits.