The EGFR (also recognized as ErbB1 or HER1) belongs to the ERBB family of RTKs that consists of three additional members including ErbB2/HER-2, ErbB3/HER-3, and ErbB4/HER-4 (37, 38). The EGFR emerges as a typical transmembrane receptor that triggers signaling cascades through ligand-elicited dimerization and tyrosine kinase activation (39). The EGFR possesses diverse ligands such as amphiregulin (AR), betacellulin, epidermal growth factor (EGF), transforming growth factor (TGF) -α and heparin-binding EGF. Aberrant EGFR activation stimulates multiple signaling pathways that mediate cellular dysfunctions and pathologies (40, 41).

ErbB-1 undergoes tyrosine phosphorylation in response to ligand binding, whereas overexpression of c-Cbl can mitigate this effect (27, 28, 42). c-Cbl causes the ubiquitin-dependent degradation and down-regulation of ErbB-1, but not ErbB-3. Through c-Cbl’s direct binding to phosphotyrosine 1045 (pY1045) in ErbB-1’s cytosplasmic domain, c-Cbl and ErbB-1 co-localize in endosomes, targeting ErbB-1 for lysosomal degradation through a ubiquitin-dependent process (43). Similarly, overexpression of Cbl-b or Cbl-3 in Chinese hamster ovary (CHO) cells accelerates removal of overexpressed EGFR from the cell surface, leading to its endocytosis and ubiquitination. Interestingly, alternative splicing of a short peptide of Cbl-3 (Cbl-3S), with a defective SH2 domain, does not influence ubiquitination of the EGFR (43). Thus, all three mammalian members of the Cbl family are implicated in desensitization of the EGFR. Furthermore, an endophilin-CIN85-Cbl complex induces ligand-dependent endocytosis and downregulation of the EGFR (44, 45).

The PDGF family consists of four polypeptide chains that form five biologically active isoforms: PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD (46). The PDGF ligands exhibit cellular effects by binding to two tyrosine kinase receptors: PDGFR-α and PDGFR-β. PDGFR-α and PDGFR-β bind to different ligands with diverse affinities, and they have similar but different activities (47). Ligand binding promotes dimerization, autophosphorylation and activation of the tyrosine kinase domain in PDGFRs (48, 49).

Stimulation of PDGFRs facilitates phosphorylation of c-Cbl, as well as their physical interaction (29). In turn, c-Cbl overexpression accelerates ligand-induced ubiquitination and subsequent degradation of PDGFR-α and PDGFR-β, as well as inhibiting proliferation and survival dependence by PDGF (50, 51). c-Cbl is able to negatively regulate PDGFR-dependent biological functions, which requires the intact tyrosine kinase binding domain of c-Cbl (30, 31, 50). Both c-Cbl and Cbl-b impact PDGFRβ polyubiquitination and internalization of the PDGFR stimulated by its ligand. Cbl-b together with c-Cbl form a complex which also interacts with PDGFRβ after PDGF-BB stimulation. c-Cbl is unable to bind directly to PDGFRβ, indicating that Cbl-b is essential for the interaction of c-Cbl with PDGFRβ (52).

The mammalian FGF family comprises 22 members which have homologous central protein sequences and structure. FGFs interact with four high affinity cell-surface RTKs designated FGFR1-4 (53–55). Binding of FGFs to FGFRs initiates intrinsic tyrosine kinase activity and multiple downstream signaling cascades, such as RAS-MAP and PI3K-AKT, mediating a wide range of cellular responses (32, 56).

FGFR2 activation recruits c-Cbl binding to the receptor, allowing ubiquitination and proteasome degradation of FGFR2 (33). Thus, c-Cbl mediates down-regulation of FGFR2 and attenuation of FGFR2 signaling, which results in PI3K/Akt attenuation and decreased osteoblast survival triggered by FGFR2 activation. On the contrary, no direct interaction has been observed between c-Cbl and FGFR3 during ubiquitination of FGFR3 (57, 58). Interestingly, a correlation has been found between c-Cbl expression and FGFR3 activity. Overactive FGFR3 mutations yield c-Cbl overexpression, while c-Cbl does not influence FGFR3 expression and ubiquitylation (59).

NGF, a member of the neurotrophin family, is expressed in both the nervous system and peripheral organs (60). NGF/NGFR signaling contributes to the development of the nervous system, angiogenesis and inflammatory diseases. NGF exerts its effects through binding to one of its two receptors: the high-affinity receptor tropomyosin receptor kinase A (TrkA) and the low-affinity receptor p75 neurotrophin receptor (p75) (34, 61). Binding of NGF to its receptors causes receptor dimerization, autophosphorylation and activation, which enhances the phosphorylation of downstream cellular proteins and signal transduction (62).

In response to NGF stimulation, c-Cbl is capable of mediating the internalization, endosomal trafficking, ligand-induced ubiquitination and subsequent lysosomal degradation of TrkA. TrkA ubiquitination and degradation also require direct association between c-Cbl and phosphorylated TrkA (63).

HGF, also known as scatter factor (SF), is mainly expressed in is stromal cells and fibroblasts. Mesenchymal epithelial transition factor (Met) the receptor of HGF, is primarily produced by epithelial cells (64). Following stimulation with HGF, the receptor tyrosine kinase Met is auto-phosphorylated and activated in the cytoplasm, triggering intracellular signaling cascade activation (35, 65).

c-Cbl, but not its oncogenic mutants v-Cbl or 70Z/3 Cbl, targets Met for ubiquitylation and degradation to downregulate HGF/Met signaling (66). c-Cbl is recruited to Met though two mechanisms: direct interaction with the juxtamembrane domain of Met by its TKB domain, and indirect interaction through Grb2 via its proline-rich domain (67). After binding to ligand-activated Met, c-Cbl undergoes tyrosine phosphorylation, and ubiquitinates Met through recruiting the endophilin-CIN85 complex, resulting in suppression of signal transduction and biological responses. Inhibition of this complex formation is able to prevent downregulation of Met. Thereby, the endophilin-CIN85-Cbl complex is involved in ligand-dependent Met downregulation (68).

CSF-1 serves as a growth factor that participates in the regulation of proliferation, differentiation and survival of mononuclear phagocytes (69). The biological activity of CSF-1 is mediated by its high-affinity cognate receptor CSF-1R encoded by the c-fms proto-oncogene. CSF-1 induces CSF-1R dimerization, resulting in the autophosphorylation of tyrosine residues in the cytoplasmic portion of the CSF-1R, which are the binding sites for Src homology 2 (SH2) containing proteins (36, 70).

c-Cbl down-regulates the CSF-1R by targeting it for polyubiquitination and subsequent enhancement in the endocytic rate, which inhibits macrophage proliferation in response to CSF-1 stimulation (71). Further study has confirmed that activated CSF-1R leads to autophosphorylation of multiple tyrosine residues, such as Tyr973 at its carboxy-terminus. The c-Cbl TKB domain binds to activated CSF-1R at the phosphorylated residue Tyr 973, resulting in CSF-1R signaling cessation (72).

CIN85, recognized as Cbl-interacting molecule of 85 kDa, is a modular-assembled adaptor protein (73). CIN85 consists of a proline-rich region, three SH3 domains and a coiled-coil region. The SH3 domains of CIN85 bind to the distal carboxyl termini of Cbl and Cbl-b, but not to the proline-rich region. There is no association between CIN85 and Cbl-3 in mammalian cells (74, 75).

Studies have indicated that CIN85 and Cbl family interactions are involved in the regulation of activated RTKs. These associations are further enhanced upon tyrosine phosphorylation of c-Cbl or Cbl-b stimulated after EGF and PDGF activation. CIN85 binding to Cbl-b is essential for internalization of EGFR, however it has no direct influence on receptor ubiquitination stimulated by Cbl-b (44). Further, c-Cbl has been found to mediate RTK endocytosis via an interaction with the CIN85-endophilin complex. The Cbl-CIN85-endophilin association induces ligand-stimulation degradation of the EGFR and c-Met. Suppression of the Cbl-CIN85-endophilin complex formation is enough to prevent RTK endocytosis and downregulation, without affecting the ubiquitination function of c-Cbl (45, 68).

Growth factor receptor bound protein 2 (Grb2) is a widely expressed adaptor molecule, which mediates various basic cellular functions and downstream signaling pathways (76). Grb2 consists of a Src homology2 (SH2) domain flanked by N- and C-terminal SH3 domains. The Grb2 SH2 domain associates directly with the tyrosine residues in autophosphorylated EGFR, and Grb2 also binds to other RTKs, such as HGFR (77).

Proline-rich sequences of c-Cbl interact with the SH3 domains of Grb2, which indirectly recruit c-Cbl to EGFRs or Met receptors (78, 79). Grb2 negatively regulates RTKs and downstream signaling, including the Ras pathway, through the recruitment of c-Cbl (80). Tyr1045 mutant EGFR, defective in the c-Cbl docking site, shows reduced ubiquitylation and endocytosis, even under the condition of c-Cbl overexpression. Unexpectedly, an EGFR mutant defective at Tyr1045 (Y1045F) still displays ubiquitination and downregulation, most notably in the presence of c-Cbl overexpression and Grb2, because the Grb2/c-Cbl complex is recruited to Grb2 docking sites on the EGFR (78). Additionally, other studies have shown that Grb2 exerts positive effects on RTK signaling and activates the Ras pathway via its interaction with guanine nucleotide exchange factor Sos (16). Hence, Grb2 acts as a double-edged sword in its regulation of RTKs signaling.

Sprouty was first discovered in Drosophila as a new antagonist of the FGF signaling pathway. There are four Sprouty isoforms in mammals, which include Sprouty1-4 ( (81). The Sprouty proteins are identified as an additional family of putative signaling regulators. The Sprouty family is shown to inhibit RTKs, specifically by suppressing downstream Ras/Raf/ERK signaling activation induced by growth factors such as FGF, PDGF, NGF and VEGF (82, 83).

Sprouty2 can directly bind to the RING finger domain of c-Cbl, and then remove c-Cbl from activated EGFR. As a result, Sprouty2 abrogates c-Cbl-mediated EGFR internalization and ubiquitylation, thus sustaining downstream receptor signaling (84–87). Other Sprouty homologs, such as Sprouty3 and Sprouty4, do not affect EGFR degradation, although they have the c-Cbl-binding motif (80). Moreover, Sprouty2 interacts with CIN85, and functions at the Cbl/CIN85 interface following EGF stimulation, consequently blocking EGFR downregulation (88).