Figure 2.

β-Arrestin binding on azF-incorporated AT1R mutants. A and B, AngII-mediated photocross-linking of azF-incorporated AT1Rs with β-arr1. HEK293T cells transiently expressing each of the AT1R amber mutants in the absence (−) and presence (+) of 0.5 mm azF were incubated with vehicle (−) or 1 μm AngII (+) followed by exposure (+) or not (−) to UV as described under “Experimental procedures.” Total cell lysates were then immunoprecipitated (IP) using an anti-FLAG antibody to isolate AT1Rs, and immunoprecipitated proteins were resolved by SDS-PAGE. Cross-linked complexes were detected with an anti-β-arr1 antibody (immunoblot (IB)). Shown are representative blots from three to seven independent experiments. C, quantification of β-arr cross-linking (black; above x axis), expression (green; below x axis), and β-arr1 recruitment to AT1R amber mutants (orange; below x axis). Receptors were transiently transfected in HEK293T cells in the presence of 0.5 mm azF, and their expression levels were determined through detection of C-terminal RlucII epitope. β-Arr binding to receptors was determined by BRET as described under “Experimental procedures” and represents the E_max of BRET ratio. Quantifications of optical density from the blots' bands and BRET signals are the mean ± S.E. (error bars), normalized to the A225azF mutant (dashed lines). N.D., not detected. D, schematic summary of AT1R contacts with β-arr.