Fig 2: Functional divergence of β-arr isoforms at the IGF-1R. (a) The agonist-unstimulated IGF-1R has a greater affinity for β-arr2 than β-arr1, initiating a low level of Mdm2-dependent IGF-1R ubiquitination, which in β-arr2 predominance conditions leads to ligand-independent degradation. β-arr2 in the cytoplasm sequesters β-arr1 by forming biologically inactive heterodimers. (b) Ligand-induced receptor conformational changes cause a switch in affinity towards the β-arr1 isoform, mediating stable β-arr1/IGF-1R association, enhanced Mdm2-dependent ubquitination and receptor internalization. In conditions with β-arr2 predominance, β-arr2 dissociates faster from the ligand-occupied receptor, which delays ligand-dependent receptor degradation through recycling pathways. (c) A strong and prolonged β-arr1/IGF-1R interaction sustains ERK activity and eventual degradation, whereas the weaker interaction of β-arr2/IGF-1R is mirrored by transient ERK activation (d). β-arr1 signalling predominance maintains low p53 levels, allowing cell cycle progression. β-arr2 opposes this process, possibly by sequestering both β-arr1 and Mdm2 in the cytoplasm, rendering cell cycle arrest.

Fig 3: Role of β-arrestin2 in IGF-1R-mediated signalling. (a–c) MEFs knockout for either β-arr isoform (a) or MEF WT (b) and HEK293T (HEK) (c) transfected with isoform-specific β-arr2-encoding plasmid (+β2), siRNA (-β2) or respective controls (M), were serum starved and stimulated with IGF-1 (50 ng/ml) for 0–60 min. Lysates were analysed by WB for levels of phosphorylated (p) -IGF-1R, -AKT, -ERK, alongside total ERK and AKT, and GAPDH as a loading control (left panels). Late (30 and 60 min) ERK phosphorylation levels were quantified by densitometry, normalized to GAPDH and displayed as fold change from unstimulated level (graphs, right panel). Values indicate mean±s.e.m. from three independent experiments. Statistical analysis (two-tailed t-test): changes of IGF-1-dependent late pERK induced by β-arr modulation (KO, +, −) compared with their respective WT or M (empty vector/non-target siRNA) control, *P<0.05, **P<0.01, ***P<0.001.

Fig 4: Dependency on Mdm2 of the β-arr2-mediated IGF-1R degradation and signalling. (a, b) MEF expressing truncated IGF-1R, defective in binding Mdm2 (MEF 1245), transfected with isoform-specific β-arr2-encoding plasmid (+β2), siRNA (−β2) or respective control (M) were lysed and level of β-arr2 verified by WB, using GAPDH as a loading control (a, left panel). Transfected cells were serum starved and stimulated with IGF-1 (50 ng/ml) for 0, 12 or 24 h. Lysates were analysed by WB for IGF-1R, quantified by densitometry, normalized to GAPDH and displayed as percentage of mock unstimulated controls (a). Parallel samples were stimulated for 0–60 min and analysed by WB for phosphorylated (p) -IGF-1R, -AKT, -ERK and GAPDH. Late (30 and 60 min) ERK phosphorylation levels were quantified by densitometry, normalized to GAPDH and displayed as fold change from unstimulated level (graphs, right panel). Values indicate mean±s.e.m. from three independent experiments. (c, d) U2OS and SAOS2, transiently transfected with isoform-specific β-arr2-encoding plasmid (+β2), siRNA (−β2) or respective control (M) were lysed and levels of β-arr2 level verified by WB, using GAPDH as a loading control (c, left panel). Transfected cells were serum starved and stimulated with IGF-1 (50 ng/ml) for 0, 12 or 24 h. Lysates were analysed by WB for IGF-1R, quantified by densitometry, normalized to GAPDH and displayed as percentage of mock unstimulated controls (c, right panel, graphs). Parallel samples were stimulated for 0–60 min and analysed by WB for phosphorylated (p) -IGF-1R, -AKT, -ERK and GAPDH. Late (30 and 60 min) ERK phosphorylation levels were quantified by densitometry, normalized to GAPDH and displayed as fold change from unstimulated level (graphs, right panel). Values indicate mean±s.e.m. from three independent experiments (d). Statistical analysis (two-tailed t-test): IGF-1-induced degradation rate following β-arr modulation (+, −) compared with their respective M (empty vector/non-target siRNA) controls (a, c). Changes of IGF-1-dependent late pERK induced by β-arr modulation (+, −) compared with their respective M (empty vector/non-target siRNA) control (b, d). *P<0.05, ***P<0.001.

Fig 5: Representative images of HPS and IHC stainings in lung ADC and SCC samples. Automated IHC was performed on sections of the in-house TMA using antibodies that recognise beta-arrestin-1-2 (ARRB1-2), the commonly used ADC markers (TTF1, NAPSA, KRT7) and SCC markers (KRT5-6 and p63). DAB-based visualisation was used to assess protein expression. For each sample, a haematoxylin/phloxine/saffron (HPS)-stained slide is shown