The insulin-like growth factor-1 receptor (IGF-1R) plays important roles in physiological growth and aging as well as promoting several crucial functions in cancer cells. However, the molecular mechanisms involved in expression and down-regulation of IGF-1R are still poorly understood. Here we provide evidence that -arrestin, otherwise known to be involved in the regulation of G protein-coupled receptors, serves as an adaptor to bring the oncoprotein E3 ubiquitin ligase MDM2 to the IGF-1R. In this way, -arrestin acts as a crucial component in the ubiquitination and down-regulation of the receptor. Both MDM2 and -arrestin co-immunoprecipitated with the IGF-1R. The -arrestin isoform 1 appeared to be more strongly associated with the receptor than isoform 2, and in a molecular context it was 4-fold more efficient in inducing polyubiquitination of IGF-1R, a reaction that required the presence of -arrestin and MDM2. Ligand stimulation accelerated IGF-1R ubiquitination. In mouse P6 cells (overexpressing human IGF-1R) absence of -arrestin 1, but not of -arrestin 2, blocked ubiquitination of IGF-1R. Conversely, in the two studied human melanoma cell lines both -arrestin isoforms seemed to be involved in IGF-1R ubiquitination. However, because depletion of -arrestin 1 almost completely eliminated degradation, and IGF-1 induced down-regulation of the receptor in these cells, whereas -arrestin 2 only had a partial effect, -arrestin 1 seems to the more important isoform in affecting the expression of IGF-1R. To our knowledge this is the first study demonstrating a defined molecular role of -arrestin with direct relevance to cell growth and cancer.
-Arrestin1, which regulates many aspects of seven transmembrane receptor (7TMR) biology, has also been shown to serve as an adaptor, which brings Mdm2, an E3 ubiquitin ligase to the insulin-like growth factor-1 receptor (IGF-1R), leading to its proteasome-dependent destruction. Here we demonstrate that IGF-1R stimulation also leads to ubiquitination of -arrestin1, which regulates vesicular trafficking and activation of ERK1/2. This -arrestin1-dependent ERK activity can occur even when the classical tyrosine kinase signaling is impaired. siRNA-mediated suppression of -arrestin1 in human melanoma cells ablates IGF-1-stimulated ERK and prolongs the G 1 phase of the cell cycle. These data suggest that -arrestin-dependent ERK signaling by the IGF-1R regulates cell cycle progression and may thus be an important regulator of the growth of normal and malignant cells.
BackgroundThe insulin-like growth factor 1 receptor (IGF-1R) plays numerous crucial roles in cancer biology. The majority of knowledge on IGF-1R signaling is concerned with its role in the activation of the canonical phosphatidyl inositol-3 kinase (PI3K)/Akt and MAPK/ERK pathways. However, the role of IGF-1R ubiquitination in modulating IGF-1R function is an area of current research. In light of this we sought to determine the relationship between IGF-1R phosphorylation, ubiquitination, and modulation of growth signals.MethodologyWild type and mutant constructs of IGF-1R were transfected into IGF-1R null fibroblasts. IGF-1R autophosphorylation and ubiquitination were determined by immunoprecipitation and western blotting. IGF-1R degradation and stability was determined by cyclohexamide-chase assay in combination with lysosome and proteasome inhibitors.Principal FindingsIGF-1R autophosphorylation was found to be an absolute requirement for receptor ubiquitination. Deletion of C-terminal domain had minimal effect on IGF-1 induced receptor autophosphorylation, however, ubiquitination and ERK activation were completely abolished. Cells expressing kinase impaired IGF-1R, exhibited both receptor ubiquitination and ERK phosphorylation, however failed to activate Akt. While IGF-1R mutants with impaired PI3K/Akt signaling were degraded mainly by the proteasomes, the C-terminal truncated one was exclusively degraded through the lysosomal pathway.ConclusionsOur data suggest important roles of ubiquitination in mediating IGF-1R signaling and degradation. Ubiquitination of IGF-1R requires receptor tyrosine kinase activity, but is not involved in Akt activation. In addition we show that the C-terminal domain of IGF-1R is a necessary requisite for ubiquitination and ERK phosphorylation as well as for proteasomal degradation of the receptor.
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