Our results can be summarized as follows. (i) In mouse embryo fibroblasts overexpressing Grb10 and the IGF-IR (p6/Grb10), there is a strong ligand-dependent increase in ubiquitination of the IGF-IR compared with that in p6 cells. (ii) Ubiquitination of the IGF-IR is associated with a shorter half-life and increased internalization of the receptor in p6/Grb10 cells. (iii) The IGF-IR is stabilized following treatment with both MG132 and chloroquine, indicating that both the proteasome and lysosomal pathways mediate degradation of the receptor. (iv) Ubiquitination of the IGF-IR occurs at the plasma membrane or at a very early stage in vesicle formation, as it is insensitive to dansylcadaverine, a specific inhibitor of early endosomes in IGF-IR trafficking. (v) Grb10 coimmunoprecipitates in vivo with the IGF-IR and endogenous Nedd4, suggesting the presence of a complex in p6/Grb10 cells. (vi) Ubiquitination of the IGF-IR in p6/Grb10 cells is impaired by overexpression of Nedd4(CS), which also stabilizes the receptor. (vii) Overexpression of a Grb10 mutant lacking the SH2 domain impairs IGF-I-mediated ubiquitination of the IGF-IR in both p6 and p6/Grb10 cells, suggesting that binding between Grb10 and Nedd4 is critical for ubiquitination of the receptor.
The adapter protein Grb10 belongs to a superfamily of related proteins including Grb7, -10, and -14 and C. elegans
Mig10 (reviewed in reference 35
). Grb10 was isolated in our and other laboratories as an interacting partner of the IGF-IR or the IR (35
). Previous and recent work has shown an inhibitory role for mGrb10α in IGF-I-mediated mitogenesis (38
), and mGrb10α was used as bait in a yeast two-hybrid screen to isolate mouse Nedd4, a ubiquitin protein ligase, as an interacting protein of Grb10 (36
Nedd4 contains a C2 domain, three or four WW domains and a ubiquitin ligase HECT domain. The WW domains mediate binding to target proteins, usually by associating with their PY motifs (PPxY) (23
). The IGF-IR does not have PY motifs, suggesting that there is no direct interaction between these two proteins. In fact, when we used the two-hybrid system we did not detect any direct binding between the IGF-IR and Nedd4 (data not shown). The interaction between the C2 domain of Nedd4 and the SH2 domain of Grb10, via a phosphotyrosine-independent association, did not result in ubiquitination of Grb10 (36
). It was therefore reasonable to speculate that this interaction may instead serve to localize Nedd4 to targets of Grb10 such as the IGF-IR. Indeed, ubiquitination of the IGF-IR was demonstrated previously (52
), and we now present evidence for a strong ligand-dependent in vivo ubiquitination of the IGF-IR in p6/Grb10 cells compared with parental p6 cells. This increased ubiquitination is associated with a shorter half-life and decreased levels of cell surface IGF-IR in p6/Grb10 cells. These results suggest that the ligand-mediated increased ubiquitination of the IGF-IR in p6/Grb10 cells is associated with an increase in the internalization rate for the receptor, as previously reported for the EGFR (28
). The IGF-IR in p6/Grb10 cells is likely ubiquitinated at the plasma membrane, as it occurs in the presence of dansylcadaverine, a specific inhibitor of IGF-IR endocytosis (7
). Grb10 coprecipitates in vivo with the IGF-IR and endogenous Nedd4, indicating the presence of a complex in p6/Grb10 cells. These data strongly suggest that Grb10 plays an adapter role in Nedd4-mediated ubiquitination of the IGF-IR. Significantly, it was recently shown in Drosophila
that DNedd4 binds Commissureless, and Commissureless acts as an adapter protein to target Roundabout for internalization in a DNedd4-dependent manner (40
). Moreover, Cbl-induced ubiquitination of the T-cell receptor ζ requires the adapter role of Zap-70 (61
), pointing to a possible common alternative molecular mechanism that requires the presence of an adapter protein in E3-mediated ubiquitination of receptor proteins.
p6/Grb10 cells are severely impaired in IGF-I-stimulated cell proliferation compared with parental p6 cells (38
), implicating Grb10 in this impairment. Accordingly, our results suggest that Grb10, by recruiting Nedd4, promotes increased ubiquitination, faster internalization, and subsequent enhanced degradation of the IGF-IR in p6/Grb10 cells, thus negatively regulating IGF-IR downstream signaling and mitogenesis. This is reminiscent of the negative role of Cbl, a RING finger E3, in mitogenesis via receptor tyrosine kinases, such as the EGFR, platelet-derived growth factor receptor, colony-stimulating factor 1 receptor, and Met (27
). However, we cannot rule out the possibility that Grb10 could also inhibit IGF-IR signaling by additional mechanisms, like direct inhibition of the kinase activity of the receptor by binding of the Grb10 BPS region, as recently reported by other investigators (5
Our results show that specific inhibitors of both the proteasome and lysosomal pathways stabilize the IGF-IR protein, indicating that both pathways contribute to the degradation of the receptor. There is precedence for degradation of transmembrane proteins by both pathways. For example, in ENaC, the unassembled chains are targeted to the proteasome while the assembled channel is degraded by the lysosome and possibly also by the proteasome (54
). Moreover, although many cell surface proteins, particularly in Saccharomyces cerevisiae
, are degraded by the lysosome and/or vacuole, proteasome involvement has been demonstrated in several others, such as the growth hormone receptor (59
), LMP1 (2
), and several tyrosine kinase receptors (21
Overexpression of the Nedd4(CS) mutant impaired ubiquitination of the IGF-IR in p6/Grb10 cells (Fig. ) and partially stabilizes the receptor (Fig. ), indicating that the mutant Nedd4 acts in a dominant negative fashion towards the endogenous Nedd4. The effect of Nedd4(CS) on stability of the IGF-IR is not as pronounced as in inhibiting ubiquitination. This result can be explained given that (i) endogenous Nedd4 is expressed at high levels in p6/Grb10 cells (36
) and that (ii) in the ubiquitination experiments Nedd4(CS) competes with the endogenous Nedd4 for ubiquitination of only a fraction of total receptor (the portion that is ubiquitinated), while in pulse-chase experiments the Nedd4(CS) mutant acts on the total pool of newly synthesized IGF-IR. These results therefore suggest that Nedd4 is likely the E3 ligase involved in the regulation of ubiquitination, sorting, and stability of the IGF-IR in p6/Grb10 cells. We cannot rule out the possibility, however, that other ubiquitin ligases could also participate in the ubiquitination of the IGF-IR, utilizing mechanisms independent of Grb10.
Ligand-dependent internalization through clathrin-coated pits and subsequent degradation of tyrosine kinase receptors is a critical step in modulating their biological activity (9
). But while the role of the ubiquitin ligase Cbl is well established in tyrosine kinase receptor ubiquitination and regulation (27
), our data represent, to our knowledge, the first evidence for the involvement of the Nedd4 (HECT) family of E3 ubiquitin ligases in the regulation of ubiquitination, internalization, and stability of tyrosine kinase receptors. Moreover, unlike the regulation of ENaC by direct binding of Nedd4 family members, regulation of the IGF-IR by Nedd4 employs an adapter protein, Grb10 (Fig. ). Furthermore, this Nedd4/Grb10 complex formation is not dependent on Nedd4-WW binding to PY motifs on target proteins, as the interaction is mediated by the SH2 domain of Grb10 and the C2 domain of Nedd4 (36
). Significantly, the expression in parental p6 and p6/Grb10 cells of the ΔSH2 mutant inhibits IGF-I-mediated ubiquitination of the IGF-IR (Fig. ), confirming that the binding of endogenous and exogenously expressed Grb10 to Nedd4 is critical for ubiquitination of the receptor.
FIG. 9. Model for the role of Grb10 as an adapter connecting Nedd4 to the IGF-IR. The Grb10 SH2 domain constitutively binds the C2 domain of Nedd4. Upon ligand stimulation, Grb10 binds the activated IGF-IR through the BPS domain, allowing the formation of a complex (more ...)
Nedd4 and its yeast homologue Rsp5p had been previously shown to regulate the stability of numerous cell surface proteins (reviewed in reference 51
), as well as several cytoplasmic and nuclear proteins (4
). More recently, Nedd4 members were shown to also regulate ubiquitination involved in vesicular transport and sorting (16
) and virus particle budding (18
). Our work extends our understanding of Nedd4 function, i.e., regulation of ubiquitination and stability of receptor tyrosine kinases, by use of the adapter protein Grb10 and possibly other family members, such as Grb7 and Grb14 (30
), which also bind Nedd4.