Figure 1.
Wild type and mutant IGF-1R constructs.
A. IGF-1R is a heterodimeric receptor with two identical ß-subunit and two identical α-subunits. The α-subunit is completely extracellular, while the ß-subunit spans the membrane. The intracellular portion (residues 931–1337) has a TK domain with intrinsic kinase activity. Important tyrosine residues in the TK domain, know as activation loop, are (Y1131, Y1135 and Y1136) shown in green. Tyrosine residue Y950 (in blue) is, when phosphorylated, a docking site for binding of signaling molecules. Other important regions of the receptor are the ATP binding site (K1003) and the C-terminal domain important for phosphorylation and signaling, respectively. B. The expression of all IGF-1R variants were investigated on cells growing under basal conditions (complete medium supplemented with serum). Cell lysates were subjected to western blotting using antibodies to IGF-1R (β-subunit) and GAPDH (loading control).
Figure 2.
Ubiquitination of IGF-1R is phosphorylation dependent.
After 24-h serum starvation wt IGF-1R (A) and K1003R (B) cells were stimulated for indicated time points with 50 ng/ml IGF-1. Lysates were immunoprecipitated (IP) with anti-IGF-1Rß (H60), and blotted with either anti-phosphotyrosine (pY99) or anti-ubiquitin (p4D1). The graphs represent quantification (% of loading control) of three independent experiments. Means and SDs are shown. Equal loading was confirmed by stripping and re-immunoblotting with anti-IGF-1Rß (C20).
Figure 3.
C-terminal domain is essential for ubiquitination of IGF-1R.
After 24-h serum starvation Y1136, Y950F, Δ1245 and Y950F-Δ1245 cells were stimulated for the indicated time points with IGF-1. Determinations of IGF-1R phosphorylation and ubiquitination were performed as described in Fig. 2. The investigated signals from three independent experiments were quantified (% of loading control) and presented in graphs above each blot. Means and SDs are shown.
Figure 4.
Ubiquitination of IGF-1R is crucial for ERK signaling.
After 24 h serum starvation wt, Y1136F, K1003R, and Δ1245 cells were stimulated with IGF-1 for the indicated times and lyzed. The lysates were resolved by SDS-PAGE and analyzed by western blotting with anti-phospho-Akt, anti-phospho-ERK. The blots were then stripped and reprobed with anti-Akt, anti-Erk antibodies to demonstrate equal loading. The experiments were repeated with similar results.
Figure 5.
IGF-1R is degraded through both proteasomes and lysosomes.
A. Wt, K1003R, and Δ1245 were either untreated (Unt) or pre-incubated with epoxomicin (PI) or chloroquine (LyI) as described in Materials and methods. Cycloheximide (CHX) (50µg/ml) was added and the cells were further incubated for 6 or 12 h. The cells were lyzed and subjected to SDS-PAGE and western blotting with antibody to IGF-1Rß. The proreceptor and 95 kDa β-subunit of IGF-1R is indicated. B shows quantified data of IGF-1R, as normalized with GAPDH. Means and SDs of three separate experiments are shown. * P,<0.05; **P,<0.005.
Figure 6.
Intact Y1136 and Y950 are important for lysosomal degradation of IGF-1R.
Y1136F, Y959F, and Y950F-Δ1245 cells were pre-incubated without or with PI or LyI and treated with CHX as described in Fig. 5. After western blotting quantification of IGF-1R, as normalized with GAPDH, was performed. Means and SDs of three different experiments are shown. *P,<0.05; ** P,<0.005.