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.

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.

mAbs to receptor tyrosine kinases such as EGF receptor͞ErbB-1 and HER2͞ErbB-2 inhibit the tumorigenic growth of certain cancer cells, but although recombinant versions of such Abs are already used in oncology wards, the mechanism underlying immunotherapy remains unknown. We report that anti-EGF receptor Abs promote a slow endocytic process distinct from the rapid EGF-induced receptor internalization. Combining mAbs that engage distinct epitopes significantly accelerates receptor degradation. In addition, mAb combinations are more effective than single Abs in inhibiting HER2 signaling in vitro and tumorigenesis in animals. We present a model attributing efficacy of immunotherapy to the size of Abreceptor lattices formed at the cell surface, which dictates the rate of endocytic clearance and extent of signaling blockade.ErbB ͉ growth factor ͉ oncogene ͉ signal transduction ͉ antibody T he four receptor tyrosine kinases of the ErbB family and their multiple ligand molecules form a layered signaling network, which is implicated in human cancer (reviewed in ref.1): overexpression of ErbB-1͞EGF receptor (EGFR) has been implicated as a feature of poor prognosis in various human malignancies. Moreover, deletion mutants of EGFR exist in brain tumors and point mutations have recently been reported in lung cancer (2). By contrast, ErbB-2͞HER2 is rarely mutated in solid tumors. Instead, the erbB-2 gene is frequently amplified in breast, ovarian, and lung cancer (3). Because of their oncogenic potential and accessibility, ErbB proteins have emerged as attractive targets for pharmaceutical interventions. One major strategy involves the use of mAbs. Early studies uncovered the tumor-inhibitory potential of mAbs directed at ErbB-1 and ErbB-2 (4, 5), and later studies indicated that anti-ErbB mAbs are effective when combined with various chemotherapeutic agents (6, 7). Indeed, the clinical benefit of combining mAbs with certain chemotherapeutic agents was notable, which led to the approval of mAbs to ErbB-2 (Herceptin) and EGFR (C225͞ Cetuximab) for the treatment of breast and colorectal cancer, respectively.Two types of mechanisms have been implicated in ErbBdirected immunotherapy. The first involves mAb-mediated recruitment to tumors of natural killer cells through the Fc-␥ activation receptors of these immune effector cells (8). The second type of mechanisms relates to intrinsic mAb activities, which include blockade of ligand binding or receptor heterodimerization (9), inhibition of downstream signaling to Akt (10), and acceleration of receptor internalization (11,12). The latter mechanism is particularly attractive because ligand-induced endocytosis and degradation of active receptor tyrosine kinases (RTKs) is considered a major physiological process underlying attenuation of growth-promoting signals (13).Several studies reported cooperative effects of mAb combinations (9, 12, 14-16), whereas others found that bivalent, Fc-lacking versions of anti-ErbB mAbs inhibit tumorigenic growth in animals (17, 18). These observations ...

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