Cells were selected in the presence of G418 and sorted. (A) Ba/F3 cells were electroporated with PDGFRB (WT, V665A or N666K) or an empty vector. P.V665A mutant receptor does not stimulate cell proliferation. Membranes were re‐probed with anti‐PDGFRB and anti‐Actin antibodies. The receptor phosphorylation was probed with an anti‐phospho‐tyrosine antibody. Total cell lysates were analysed by Western blotting. As positive control, cells expressing the WT receptor were stimulated with PDGF‐BB for 15 min. (C) HEK293T cells were transfected with WT or mutant PDGFRB or the empty vector and starved for 7 h before lysis. Statistical analysis was performed using an anova one‐way test with Dunnett correction to compare mutant to wild‐type conditions. The mean of four independent experiments is shown with SEM. As positive control, cells expressing the WT receptor were stimulated with PDGF‐BB. Results were normalized using a renilla luciferase control reporter. γ2A (A) or HEK293T (B) cells were co‐transfected with wild‐type (WT) or mutant PDGFRB and a firefly luciferase reporter controlled by STAT‐response elements (A) or serum‐response elements (B), respectively. The activity of the mutants was analysed in a dual‐luciferase reporter assay. PDGFRB p.V665A mutant is constitutively active. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd. PDGFRB Penttinen syndrome STAT1 ageing congenital disease imatinib platelet-derived growth factor targeted therapy tyrosine kinase inhibitors. In conclusion, the p.Val665Ala variant confers unique features to PDGF receptor β compared with other characterized gain-of-function mutants, which may in part explain the particular set of symptoms associated with Penttinen syndrome. Dasatinib, nilotinib and ponatinib also inhibited the mutant receptor. Importantly, this concentration remained in the therapeutic range. Another tyrosine kinase inhibitor, imatinib, blocked signalling by the p.Val665Ala variant at a higher concentration compared with the wild-type receptor. STAT1 activation was not sensitive to ruxolitinib and did not rely on interferon-JAK2 signalling. It was devoid of oncogenic activity in two cell proliferation assays, contrasting with classical PDGF receptor oncogenic mutants. Phosphorylation of STAT3, STAT5, AKT and phospholipase Cγ was weak or undetectable. In the absence of ligand, the mutant activated STAT1 and elicited an interferon-like transcriptional response. We observed that the mutant receptor was expressed at a lower level but showed constitutive activity. This substitution is located in a conserved loop of the receptor tyrosine kinase domain. The goal of the present study was to characterize the PDGFRB p.Val665Ala variant associated with Penttinen syndrome at the molecular level. Activating mutations in PDGFRB have been associated with other human diseases, including Kosaki overgrowth syndrome, infantile myofibromatosis, fusiform aneurysms, acute lymphoblastic leukaemia and myeloproliferative neoplasms associated with eosinophilia. Penttinen syndrome is a rare progeroid disorder caused by mutations in platelet-derived growth factor (PDGF) receptor beta (encoded by the PDGFRB proto-oncogene) and characterized by a prematurely aged appearance with lipoatrophy, skin lesions, thin hair and acro-osteolysis.
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