A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling

Abstract

Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c-Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c-Cbl, here we report that defective Tyr1045 of EGFR, an inducible c-Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand-induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization-defective mutant receptor imply that c-Cbl-mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand-induced ubiquitylation, especially in the presence of an overexpressed c-Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c-Cbl complex to Grb2-specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well-characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c-Cbl-dependent sorting of active tyrosine kinases to destruction.

Fig. 1. A mutant EGFR defective at Tyr1045 elicits potent signals and is refractory to c-Cbl. (A) Sublines of 32D cells that express either a wt-EGFR (squares), or a Tyr1045 mutant (Y1045F; triangles) were deprived of IL-3 and plated at a density of 5 × 10⁵ cells/ml in media containing serial dilutions of EGF. The MTT assay was performed 24 h later. The results obtained are presented as fold induction relative to a control culture maintained in the absence of added growth factor. (B) The indicated sublines of 32D cells (5 × 10⁵ cells/ml) were incubated for various time intervals with EGF at 100 ng/ml (circles). Control cultures were incubated in the absence (squares) or presence of IL-3 (triangles). Cell growth was determined daily by using the colorimetric MTT assay, and compared with the signal observed at time zero. The data presented are the mean ± SD of four determinations. (C) CHO cells were transiently transfected with plasmids encoding either a wt-EGFR (WT) or a Tyr1045 mutant (Y1045F). Alongside, we used a vector encoding c-Cbl or a control empty vector. Cell monolayers were stimulated with EGF (100 ng/ml) for the indicated time intervals at 37°C. Whole-cell lysates were analyzed with an antibody specific to the active form of MAPK. (D) CHO cells were co-transfected in triplicates as in (C), along with a reporter plasmid (SRE-luc). Thirty-six hours later cells were untreated or treated with EGF (20 ng/ml). Following an additional 12 h, cells were harvested for a luciferase assay. Signals obtained were normalized to protein concentrations and are presented as average ± SD.

Fig. 2. The c-Cbl docking site of EGFR is necessary for ligand-induced receptor endocytosis and for translocation of c-Cbl to endocytic vesicles. CHO cells transiently expressing HA-tagged c-Cbl along with a wild-type GFP–EGFR (upper panel), or a similar fusion protein containing a mutation at Tyr1045 (Y1045F; lower panel), were grown on cover slips. Cells were incubated for 15 min at 37°C without or with EGF (100 ng/ml). To visualize c-Cbl, cells were fixed, permeabilized and incubated with an anti-HA antibody, followed by incubation with a Cy3-conjugated secondary antibody (red, middle column). The GFP–EGFR fluorescence is represented in the left column (green). The right column presents the overlay of GFP and Cy3 fluorescence, generating a yellow color in areas of co-localization.

Fig. 3. The c-Cbl docking site of EGFR is essential for rapid ligand endocytosis and degradation, and it enables sequestration from the recycling pathway. (A) CHO cells transiently expressing a wt-EGFR (squares), a kinase-defective mutant receptor (circles) and a Tyr1045 mutant (Y1045F; triangles) were incubated at 37°C with a radiolabeled EGF (2 ng/ml). At the indicated times, cell monolayers were acid-washed to remove surface-bound EGF. Radioactivity present in the acidic fraction was quantified in triplicates and designated surface-associated ligand. The remaining cell-associated radioactivity (internalized) was similarly quantified following cell solubilization. The ratio obtained at each time point is presented (average ± SD). (B) Monolayers of CHO cells transfected as in (A) were incubated for 1 h at 20°C with a radiolabeled EGF. Sister monolayers were pre-incubated for 30 min with chloroquine (0.2 mM; open symbols). Thereafter, cells were washed and maintained at 37°C for the indicated time intervals. Media were then collected, and the cells were solubilized. The trichloroacetic acid-soluble radioactivity was determined and the fraction of degraded ligand presented. (C) CHO cells transiently expressing wt-EGFR (squares), the Y1045F mutant receptor (circles) or a kinase-defective mutant (triangles) were allowed to internalize a radiolabeled EGF (1 ng/ml) for the indicated time periods. The remaining surface bound ligand was removed by mild acid-washing. The EGF-loaded cells were then incubated with an unlabeled EGF at 4°C, followed by 1 h at 37°C. Intact radioactive ligand was harvested from the medium following a 1 h chase period. Similarly, the fraction of surface-bound radioactivity was determined and the combined fractions designated recycled ligand. The fraction of recycled ligand is presented as average ± SD (bars) of triplicate determinations.

Fig. 4. An alternative receptor ubiquitylation pathway independent of Tyr1045 may involve Grb2. (A) CHO cells were transfected with plasmids encoding a wt-EGFR (WT) or the Y1045F mutant, along with vectors encoding c-Cbl and an HA-tagged ubiquitin. Following 48 h, cell monolayers were treated for 10 min at 37°C without or with EGF (100 ng/ml) and cell lysates subjected to immunoprecipitation (I.P.) and immunoblotting (I.B.) with the indicated antibodies. Whole-cell lysates were also analyzed (lower panel). (B) wt-EGFR or the Y1045F mutant receptor were immunopurified from transfected HEK-293 cells. Receptor immunoprecipitates were subjected to an in vitro ubiquitylation assay with a radiolabeled ubiquitin. c-Cbl, Grb2 or a combination of the two proteins was added to the reaction mixtures in the form of GST fusion proteins. Receptor immunoprecipitates were resolved by electrophoresis and proteins transferred to filters, which were first autoradiographed (upper panel, ¹²⁵I-Ub) and then immunoblotted with anti-EGFR antibodies (lower panel). (C) Immuno precipitates of Y1045F were subjected to an in vitro ubiquitylation assay in the presence of one or two of the indicated GST fusion proteins. As a control, GST was added alone (Cont.) or it was omitted from the reaction (unlabeled lane).

Fig. 5. Grb2 enhances c-Cbl-dependent ubiquitylation and degradation of EGFR in living cells. (A) Monolayers of CHO cells were transiently transfected with expression vectors encoding the indicated forms of EGFR. Alongside, plasmids encoding c-Cbl, Grb2, HA-ubiquitin and control empty vectors were used as indicated. Cell monolayers were treated for 10 min at 37°C without or with EGF (100 ng/ml). Subsequently, cell lysates were directly subjected to immunoblotting (I.B.; panels labeled NONE). Alternatively, EGFR was first isolated, and the immunoprecipitates analyzed with the indicated antibodies. (B) CHO cells were transfected with plasmids encoding the indicated forms of EGFR, along with plasmids encoding Grb2 and c-Cbl. A control culture was treated with an empty expression vector. Following stimulation with EGF (100 ng/ml; 10 min at 37°C), EGFR was isolated from cell lysates and the immuno-complexes analyzed by using antibodies to either Grb2 or EGFR.

Fig. 6. Grb2 accelerates internalization and down-regulation of the Y1045F mutant EGFR. (A) CHO cells were transfected with expression vectors encoding wt-EGFR (WT) or the Y1045F mutant. Alongside we used plasmids encoding c-Cbl (closed circles), Grb-2 (open squares), a combination of Grb2 and c-Cbl (closed squares) or an empty control vector (open circles). Forty-eight hours post-transfection, cultures were incubated at 37°C with EGF (25 ng/ml) for the indicated periods of time. Cell-bound ligand was removed, and the level of surface receptors was determined by binding of a radiolabeled EGF at 4°C. The average ± SD (bars) of triplicate determinations is shown for each time point. (B) Monolayers of CHO cells were transfected as in (A) with vectors driving expression of the indicated version of EGFR. After 48 h, cells were incubated at 37°C with a radiolabeled EGF (2 ng/ml). At the indicated time points, monolayers were acid-washed to remove surface-bound ligand. Radioactivity present in the acidic fraction was designated surface-associated ligand. The remaining cell-associated radioactivity was determined in triplicates following cell solubilization and designated internalized ligand. Symbols are as in (A). (C) CHO cells transiently overexpressing HA-tagged c-Cbl and histidine-tagged Grb2, along with a GFP–EGFR, or a fusion protein containing a mutation at Tyr1045 (Y1045F), were grown on cover slips for 48 h after transfection. Thereafter, cells were incubated for 15 min at 37°C without or with EGF (100 ng/ml). To visualize Grb2, cells were fixed, permeabilized, and incubated with a rabbit anti-His₆ followed by a Cy3-conjugated secondary antibody (red). The GFP–EGFR fluorescence is shown in the left column (green). The right column presents the overlay of GFP and Cy3 fluorescence; a yellow color is seen in areas of co-localization.

Fig. 7. Subdomains of c-Cbl involved in receptor ubiquitylation. (A) CHO cells were transfected with plasmids encoding EGFR, Grb2, the indicated HA peptide-tagged mutants of c-Cbl or a control empty vector. Whole-cell extracts were analyzed by immunoblotting either directly or after immunoprecipitation of c-Cbl. The following mutants of c-Cbl were used: a protein the SH2 domain of which is defective (G306E); and deletion mutants containing either the N-terminal half [Cbl-N(RF), residues 1–429] or the C-terminal half (Cbl-C, residues 338 to end). (B) Monolayers of CHO cells were transiently transfected with the Y1045F mutant of EGFR, along with wild type (WT) or the 70Z mutant of c-Cbl, in the presence or absence of a Grb2 expression vector. Receptor ubiquitylation was detected using a HA-tagged ubiquitin expression vector. Forty-eight hours post-transfection, monolayers were incubated without or with EGF (100 ng/ml). Cell lysates were subjected to immunoprecipitation (I.P.) with an anti-EGFR antibody and immunoblotting (I.B.) with either an antibody to HA-ubiquitin or an antibody recognizing EGFR. (C) CHO cells were co-transfected with vectors driving expression of the indicated c-Cbl mutants [see (A)], along with a plasmid encoding the Y1045F mutant receptor. As indicated, transfections were performed with a plasmid encoding Grb2 or a control empty vector. All transfections were carried out in the presence of a plasmid directing expression of a HA-tagged ubiquitin. Cells were treated for 10 min at 37°C without or with EGF (100 ng/ml). Whole-cell lysates were analyzed by immunoblotting (I.B.) either directly (panels labeled NONE) or following immunoprecipitation (I.P.).