FIGURE 4:

V₂R^ΔICL3-βarr1 complex binds clathrin, and V₂R^ΔICL3 undergoes efficient endocytosis. (A) A coIP experiment reveals that V₂R^ΔICL3-βarr1 complex (partially engaged) is able to interact with GST-clathrin-TD, similar to V₂R^WT-βarr1 complex (fully engaged). Here ScFv30 is used to stabilize V₂R-βarr1 complexes to avoid any potential clash with clathrin binding. Purified GST is used as a negative control. A representative image of four independent experiments. (B) Densitometry-based quantification of coIP data in A. Data are normalized with respect to clathrin interaction with V₂R^WT-βarr1 complex (treated as 100%). (C) Agonist-induced endocytosis of V₂R^WT and V₂R^ΔICL3 are comparable, suggesting that the inability of V₂R^ΔICL3 to engage the core interaction with βarr does not affect receptor endocytosis. Here HEK-293 cells expressing either V₂R^WT or V₂R^ΔICL3 were stimulated with 100 nM AVP (agonist) for indicated times, and the loss of surface receptor as measured by reactivity of HRP-coupled M2 antibody with N-terminal FLAG tag in whole-cell ELISA was used as a readout of receptor endocytosis. Data represent average ± SEM of six independent experiments each carried out in duplicate.