Fig. 3.

US28 constitutively activates VEGF promoter. (A) In transiently transfected COS-7 cells, HA-US28-WT induced a dose-dependent constitutive activation of the VEGF gene promoter. (B) In the VEGF-reporter gene assay, only the N terminus deleted Δ2–22-US28 mutant induced a VEGF promoter activation similar to the WT receptor, whereas the G protein-uncoupled US28-R¹²⁹A mutant showed no VEGF promoter activation. (C) G protein-coupled receptor kinase 2 dominant negative GRK2-K²²⁰R and Gα_transducin (Gα_t) were cotransfected in different ratios with US28 (scavenger:US28 ratios were 0.5, 1, 2, and 4) into COS-7 cells and showed a dose-dependent VEGF inhibition, involving both Gα_q/11 and βγ subunits. (D) The use of kinases inhibitors UO126 (1 μM) and SB203580 (2 μM) revealed that MAPKs p44/42 and p38, respectively, are independently involved in US28-mediated VEGF gene promoter activation. (E) VEGF promoters of different lengths possess different binding sites for transcription factors (AP-2, activator protein-2; Sp1, stimulating protein 1; HRE, hypoxia inducible factor-1 responsive element; STAT3, signal transducer and activator of transcription 3). The US28-mediated VEGF promoter activation was related to the length of the promoters, highlighting the involvement of transcription factors such as the hypoxia inducible factor-1 (HIF-1), STAT3, AP-2, and Sp1.