Epidermal Growth Factor Receptor Endocytosis Research Articles

Human mammary epithelial cells rapidly exchange empty EGFR between surface and intracellular pools.

Binding of ligand to the epidermal growth factor receptor (EGFR) initiates a series of processes including activation of the intrinsic EGFR tyrosine kinase, receptor autophosphorylation, and the assembly of active signaling complexes at the plasma membrane. Concomitantly, receptor trafficking is initiated, and the receptor is ultimately delivered to the lysosome, where it is degraded. Virtually all studies on EGFR trafficking have used fibroblasts and transformed cells. Because EGFR exerts a potent effect on the physiology of epithelial cells, we examined the regulation of EGFR activity and trafficking in nontransformed human mammary epithelial cells (HMEC). We found that HMEC that displayed a luminal phenotype were largely unresponsive to EGF and maintained a majority of their EGFR at the cell surface. In contrast, HMEC with a basal phenotype were highly responsive to EGF and, at steady state in the absence of exogenous ligand, distributed empty EGFR into intracellular pools. Maintenance of the intracellular pools was a direct consequence of specific and rapid endocytosis of the empty EGFR. The trafficking pattern was EGFR specific, used coated pits, and did not require receptor tyrosine kinase activity. Such an mechanism redistributes EGFR signaling potential among different membrane domains and into vesicles with unique biochemical microenviroments. In addition, our data show that EGFR endocytosis can be regulated in the absence of ligand binding and receptor activation in a cell-type-specific manner.

Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors.

The involvement of Ral and its downstream molecules in receptor-mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO-IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full-length RalBP1 which is an effector protein of Ral, but was inhibited by its C-terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand-dependent receptor-mediated endocytosis.

Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation.

Epidermal growth factor (EGF) stimulates the homodimerization of EGF receptor (EGFR) and the heterodimerization of EGFR and ErbB2. The EGFR homodimers are quickly endocytosed after EGF stimulation as a means of down-regulation. However, the results from experiments on the ability of ErbB2 to undergo ligand-induced endocytosis are very controversial. It is unclear how the EGFR-ErbB2 heterodimers might behave. In this research, we showed by subcellular fractionation, immunoprecipitation, Western blotting, indirect immunofluorescence, and microinjection that, in the four breast cancer cell lines MDA453, SKBR3, BT474, and BT20, the EGFR-ErbB2 heterodimerization levels were positively correlated with the ratio of ErbB2/EGFR expression levels. ErbB2 was not endocytosed in response to EGF stimulation. Moreover, in MDA453, SKBR3, and BT474 cells, which have very high levels of EGFR-ErbB2 heterodimerization, EGF-induced EGFR endocytosis was greatly inhibited compared with that in BT20 cells, which have a very low level of EGFR-ErbB2 heterodimerization. Microinjection of an ErbB2 expression plasmid into BT20 cells significantly inhibited EGF-stimulated EGFR endocytosis. Coexpression of ErbB2 with EGFR in 293T cells also significantly inhibited EGF-stimulated EGFR endocytosis. EGF did not stimulate the endocytosis of ectopically expressed ErbB2 in BT20 and 293T cells. These results indicate that ErbB2 and the EGFR-ErbB2 heterodimers are impaired in EGF-induced endocytosis. Moreover, when expressed in BT20 cells by microinjection, a chimeric receptor composed of the ErbB2 extracellular domain and the EGFR intracellular domain underwent normal endocytosis in response to EGF, and this chimera did not block EGF-induced EGFR endocytosis. Thus, the endocytosis deficiency of ErbB2 is due to the sequence of its intracellular domain.

EGF Receptor Signaling Stimulates SRC Kinase Phosphorylation of Clathrin, Influencing Clathrin Redistribution and EGF Uptake

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.

ErbB-2 Amplification Inhibits Down-regulation and Induces Constitutive Activation of Both ErbB-2 and Epidermal Growth Factor Receptors

ErbB-2/HER2 is an important signaling partner for the epidermal growth factor receptor (EGFR). Overexpression of erbB-2 is also associated with poor prognosis in breast cancer. To investigate how erbB-2 amplification affects its interactions with the EGFR, we used a human mammary epithelial cell system in which erbB-2 expression was increased 7-20-fold by gene transfection. We found that amplification of erbB-2 caused constitutive activation of erbB-2 as well as ligand-independent activation of the EGFR. Overexpression of erbB-2 strongly inhibited erbB-2 down-regulation following transactivation by EGFR. Significantly, down-regulation of activated EGFR was also inhibited by erbB-2 amplification, resulting in enhanced ligand-dependent activation of the EGFR. The rate of EGFR endocytosis was not affected by erbB-2 overexpression, but the rate of lysosomal targeting was significantly reduced. In addition, erbB-2 overexpression promoted rapid recycling of activated EGFR back to the cell surface and decreased ligand dissociation from the EGFR. Our data suggest that overexpression of erbB-2 inhibits both its down-regulation and that of the EGFR. The net effect is increased signaling through the EGFR system.

Src phosphorylates EAST, a novel EGF receptor-associated protein

We have recently found and characterized EAST, a novel EGF receptor-associated protein with SH3 and TAM domains. In this study we show that EAST becomes phosphorylated by Src kinase. This, in conjunction with our earlier observations on the close association between EAST and the endocytic machinery, suggests that EAST could be involved in Src-dependent effects on EGF receptor endocytosis.

Overexpression of Cellular Src in Fibroblasts Enhances Endocytic Internalization of Epidermal Growth Factor Receptor

Previous studies have demonstrated a requirement for the nonreceptor tyrosine kinase, cellular Src (c-Src), in epidermal growth factor (EGF)-induced mitogenesis and a synergistic interaction between c-Src and EGF receptor (EGFR) in tumorigenesis. Although endocytic internalization of EGFR may be thought to attenuate EGF-stimulated signaling, recent evidence suggests that signaling through Ras can be amplified by repeated encounters of endosome-localized, receptor. Shc.Grb2.Sos complexes with the plasma membrane, where Ras resides almost exclusively. Based on these reports, we examined EGFR trafficking behavior in a set of single and double c-Src/EGFR C3H10T1/2 overexpressors to determine if c-Src affects basal receptor half-life, ligand-induced internalization, and/or recycling. Our results show that overexpression of c-Src causes no change in EGFR half-life but does produce an increase in the internalization rate constant of EGF.EGFR complexes when the endocytic apparatus is not stoichiometrically saturated; this effect of c-Src on EGFR endocytosis is negligible at high receptor occupancy in cells overexpressing the receptor. In neither case are EGFR recycling rate constants affected by c-Src. These data indicate a functional role for c-Src in receptor internalization, which in turn could alter some aspects of EGFR signaling related to mitogenesis and tumorigenesis.

Erratum: Requirement for the Adapter Protein GRB2 in EGF Receptor Endocytosis

Figure 4B (p. 1937) in the report "Requirement for the adapter protein GRB2 in EGF receptor endocytosis" by Z. Wang and M. F. Moran (28 June, p. 1935) was printed too darkly. The correct figure appears below. [See figure in the PDF file]

Requirement for the Adapter Protein GRB2 in EGF Receptor Endocytosis

Activated epidermal growth factor (EGF) receptors induce the formation of various complexes of intracellular signaling proteins that are mediated by SRC homology 2 (SH2) and SH3 domains. The activated receptors are also rapidly internalized into the endocytotic compartment and degraded in lysosomes. EGF stimulation of canine epithelial cells induced a rapid and transient association of the SH3-SH2-SH3 protein GRB2 with dynamin, a guanosine triphosphatase that regulates endocytosis. Disruption of GRB2 interactions by microinjection of a peptide corresponding to the GRB2 SH2 domain or its phosphopeptide ligand blocked EGF receptor endocytosis; other SH2 domains that bind EGF receptors or antibodies that neutralize RAS did not. Both activation and termination of EGF signaling appear to be regulated by the diverse interactions of GRB2.

Impact of receptor downregulation on clearance of two human EGFs with different receptor binding activity.

Human epidermal growth factor [hEGF(1-53)] has been thought to be cleared mainly via an EGF receptor (EGFR) endocytosis pathway. Pretreatment of rats with hEGF(1-53) has been shown previously to cause a dramatic reduction in clearance of the peptide contributable to EGFR downregulation. The impact of receptor downregulation has raised concerns for rational design of dosage regimen for this potential wound-healing therapeutic peptide. However, following a similar protocol, we could not reproduce the dramatic reduction in clearance reported previously mediated by an i.v. bolus acute dose. As EGFR downregulation may be sensitive to the length of exposure and to the activation of the receptor tyrosine kinase activity, two other pretreatment protocols were also evaluated: a 4-h i.v. infusion (prolonged exposure) of the peptide and an i.v. bolus of a potent synthetic kinase inhibitor pretreatment were evaluated for effects on clearance. However, neither pretreatment affected the peptide's clearance profile. Further, no effects on clearance and other kinetic parameters were observed for any pretreatment paradigms with a truncated analogue hEGF (1-48), whose EGF receptor binding activity is much weaker but plasma clearance is much higher than hEGF (1-53). In addition, a study in a second rat strain showed no difference in clearance profile of hEGF-(1-53) following pretreatment. Results of the present investigation suggest that receptor binding does not have a direct relationship with plasma clearance, and that the EGF clearance mechanisms is highly refractory with EGF receptors possibly recovering rapidly from downregulation through the recycling process.

Ligand-induced endocytosis of epidermal growth factor receptors that are defective in binding adaptor proteins.

Ligand-activated epidermal growth factor receptors (EGFRs) associate with coated pit adaptor proteins (AP2) in vivo, implying a mechanism for receptor retention in coated pits during internalization. Using an in vitro binding assay, we localized the adaptor binding determinant to residues 970-991 of EGFRs and confirmed specificity by competition with a synthetic peptide corresponding to this sequence. A mutant EGFR lacking this AP2 binding determinant did not associate with AP2 in vivo but demonstrated internalization and down-regulation kinetics indistinguishable from its wild-type counterpart. Immunocytochemistry confirmed ligand-induced internalization of the mutant EGFR. These data suggest that endocytic determinants are distinct from AP2 binding determinants and that processes other than association with AP2 regulate endocytosis of EGFRs.

The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.

Transforming growth factor beta 1 inhibits epidermal growth factor receptor endocytosis and down-regulation in cultured fetal rat hepatocytes

Incubation of fetal rat hepatocytes (FRH) with transforming growth factor beta 1 (TGF-beta 1) resulted in growth arrest and a biphasic effect on epidermal growth factor (EGF) receptor. After 2 h of exposure, EGF receptor (EGFR) was reduced by 43%. From 6 to 24 h, TGF-beta 1 exposure resulted in progressive increase in EGFR up to 74% over control. The increased binding was due to increase in high affinity EGF binding sites. FRH grown in medium containing EGF exhibited down-regulated EGFR with loss of high affinity EGF binding sites. With TGF-beta 1 exposure, high affinity EGFR was not down-regulated by EGF. Since down-regulation of EGFR involves internalization, the kinetics of EGF receptor-mediated endocytosis were examined. In TGF-beta 1-exposed FRH, EGF endocytosis was inhibited, with a reduction in the first order rate constant for the process from 0.078 to 0.043 min-1. Despite inhibition of growth, receptor down-regulation, and EGF endocytosis after TGF-beta 1 exposure, EGF-induced receptor autophosphorylation was preserved as demonstrated by [32P]phosphate-labeling of immunoprecipitated EGFR. These observations provide direct evidence that TGF-beta 1 regulates growth of fetal cells. Further, they suggest that TGF-beta 1 regulates endocytosis of EGF and possibly of other ligands.