Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

Ligand-induced down-regulation of two growth factor receptors, EGF receptor (ErbB-1) and ErbB-3, correlates with differential ability to recruit c-Cbl, whose invertebrate orthologs are negative regulators of ErbB. We report that ligand-induced degradation of internalized ErbB-1, but not ErbB-3, is mediated by transient mobilization of a minor fraction of c-Cbl into ErbB-1-containing endosomes. This recruitment depends on the receptor's tyrosine kinase activity and an intact carboxy-terminal region. The alternative fate is recycling of internalized ErbBs to the cell surface. Cbl-mediated receptor sorting involves covalent attachment of ubiquitin molecules, and subsequent lysosomal and proteasomal degradation. The oncogenic viral form of Cbl inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. These results reveal an endosomal sorting machinery capable of controlling the fate, and, hence, signaling potency, of growth factor receptors.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.Polypeptide growth factors regulate cellular growth by binding to surface receptors with intrinsic tyrosine kinase activity (14,54,61). These receptor tyrosine kinases constitute a family of related proteins that have been classified into subgroups on the basis of their structural homology. The receptor for epidermal growth factor (EGF), also called ErbB-1 or HER-1, is the prototype of the type I subfamily, which includes three additional members: ErbB-2/Neu, ErbB-3, and ErbB-4. Whereas ErbB-1 binds multiple distinct ligands that share the EGFlike motif (33), all of the known ligands of ErbB-3 and ErbB-4 are isoforms of the Neu differentiation factor (NDF, or neuregulin) (8,41,53), and no completely characterized ligand binds to ErbB-2 (12). Nevertheless, ErbB-2 has been implicated more than other transmembrane tyrosine kinases in cancer development (21, 50). Overexpression of this protein occurs in a significant fraction of breast and ovarian carcinomas, and it correlates with reduced patient survival (18,26,44,45). Because an oncogenic point mutation in the rat homolog of ErbB-2 mimics ligand binding (2, 59), and the ErbB-2 kinase can be stimulated by a heterologous ligand in the context of chimeric receptors (4, 30, 31), it is believed that a still unknown ligand directly binds to this orphan receptor.All members of the ErbB family are characterized by extracellular domains with two cysteine-rich sequences, and a cytoplasmic tyrosine kinase domain flanked by large hydrophilic tails, that display sequence heterogeneity and carry several tyrosine autophosphorylation sites (40, 42). The latter serve as docking sites for various cytoplasmic signaling proteins that share a 100-amino-acid-long dom...

The ErbB family includes two receptors, ErbB‐1 and ErbB‐3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB‐2. Unlike ErbB‐1 and ErbB‐2, the intrinsic tyrosine kinase of ErbB‐3 is catalytically impaired. By using interleukin‐3‐dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB‐3 is devoid of any biological activity but both ErbB‐1 and ErbB‐2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB‐3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter‐receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB‐3‐containing complexes, especially the ErbB‐2/ErbB‐3 heterodimer, are more active than ErbB‐1 complexes. Nevertheless, ErbB‐1 signaling displays dominance over ErbB‐3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen‐activated protein kinases ERK and c‐Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase‐defective ErbB‐3.

The major process that regulates the amplitude and kinetics of signal transduction by tyrosine kinase receptors is endocytic removal of active ligand^receptor complexes from the cell surface, and their subsequent sorting to degradation or to recycling. Using the ErbB family of receptor tyrosine kinases we exemplify the diversity of the down regulation process, and concentrate on two sorting steps whose molecular details are emerging. These are the Eps15-mediated sorting to clathrincoated regions of the plasma membrane and the c-Cbl-mediated targeting of receptors to lysosomal degradation. Like in yeast cells, sorting involves not only protein phosphorylation but also conjugation of ubiquitin molecules. The involvement of other molecules is reviewed and recent observations that challenge the negative regulatory role of endocytosis are described. Finally, we discuss the relevance of receptor down regulation to cancer therapy. ß

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.

Both homo-and hetero-dimers of ErbB receptor tyrosine kinases mediate signaling by a large group of epidermal growth factor (EGF)-like ligands. However, some ligands are more potent than others, although they bind to the same direct receptor. In addition, signaling by receptor heterodimers is superior to homodimers. We addressed the mechanism underlying these two features of signal tuning by using three ligands: EGF; transforming growth factor α (TGFα); and their chimera, denoted E4T, which act on cells singly expressing ErbB-1 as a weak, a strong, and a very strong agonist, respectively. Co-expression of ErbB-2, a developmentally important co-receptor whose expression is frequently elevated in human cancers, specifically potentiated EGF signaling to the level achieved by TGFα, an effect that was partially mimicked by ErbB-3. Analysis of the mechanism underlying this trans-potentiation implied that EGF-driven homodimers of ErbB-1 are destined for intracellular degradation, whereas the corresponding heterodimers with ErbB-2 or with ErbB-3, dissociate in the early endosome. As a consequence, in the presence of either co-receptor, ErbB-1 is recycled to the cell surface and its signaling is enhanced. This latter route is followed by TGFα-driven homodimers of ErbB-1, and also by E4T-bound receptors, whose signaling is further enhanced by repeated cycles of binding and dissociation from the receptors. We conclude that alternative endocytic routes of homo-and hetero-dimeric receptor complexes may contribute to tuning and diversification of signal transduction. In addition, the ability of ErbB-2 to shunt ligand-activated receptors to recycling may explain, in part, its oncogenic potential.

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both downregulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of cCbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.We have reported recently that c-Cbl can accelerate the rate of EGFR 1 degradation by increasing conjugation of polyubiquitin to an endocytosed EGFR (1). Consistent with sorting to degradation, genetic studies in Caenorhabditis elegans attributed a negative regulatory role to Sli-1, the c-Cbl ortholog of worms (2). This function is reminiscent of the action of c-Cbl downstream to the PGDFR (3), and it depends on a catalytically intact kinase domain of EGFR. The present study addressed the structural motif of c-Cbl involved in ubiquitination of EGFR. Although the 120-kDa c-Cbl protein possesses no identifiable catalytic activity, its NH 2 -terminal half carries a novel phosphotyrosine-binding domain (PYB) (4), that presumably mediates binding to the activated receptor (5). The carboxyl terminus of c-Cbl comprises several proline-rich tracts that allow constitutive binding of Src homology 3 (SH3)-containing proteins, such as Grb2, Nck, and the Cbl-associated protein, CAP (reviewed in Ref. 6). In addition, the COOH terminus is involved in inducible interactions with SH2-containing proteins, such as the regulatory subunit of the phosphoinositide 3-kinase, and the guanine nucleotide exchange protein Vav. An evolutionarily conserved RING-type zinc finger domain separates the adaptor domains of c-Cbl. RING fingers (RFs) are zinc-binding domains that differ from other zinc finger motifs in terms of structure and the zinc ligation scheme (reviewed in Ref. 7). Noteworthy is the fact that the two other family members of c-Cbl carry an intact RF (8, 9), but oncogenic Cbl variants are defective in the RF (10, 11). Our present study concentrated on the role the RF motif plays in Cbl-mediated degradation of EGFR. EXPERIMENTAL PROCEDURESMaterials, Buffers, and Antibodies-EGF was purchased from Sigma, and radioactive materials were from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). Rabbit anti-c-Cbl (C-15) antibodies, ...