FIGURE 5:

V₂R^ΔICL3-βarr1 complex binds ERK2, and V₂R^ΔICL3 exhibits efficient ERK activation. (A) V₂R^ΔICL3-βarr1 complex interacts efficiently with inactive (i.e., nonphosphorylated) and active (i.e., phosphorylated) ERK2, similar to V₂R^WT-βarr1 complex, as assessed by a coIP experiment. Here ScFv30 is used to stabilize V₂R-βarr1 complexes to avoid any potential clash with ERK2 binding. Purified GST is used as negative control. Representative image of three independent experiments. (B) Densitometry-based quantification of coIP data presented in A, showing the interaction of inactive and active ERK2 with preformed V₂R^WT/ΔICL3-βarr1 complexes. Data are normalized with respect to the signal observed for the interaction of inactive ERK2 with V₂R^WT-βarr1 complex (treated as 100%). (C) Agonist (100 nM AVP)-stimulated ERK activation in HEK-293 cells expressing V₂R^WT or V₂R^ΔICL3 as measured by Western blotting. V₂R^ΔICL3 exhibits efficient ERK activation similar to V₂R^WT, indicating that the lack of core interaction with βarr does not influence ERK phosphorylation. A representative image from six independent experiments. (D) Densitometry-based quantification of agonist-induced ERK phosphorylation in C. Data represent average ± SEM of six independent experiments normalized with respect to signal at 5 min for V₂R^WT (treated as 100%).