FIGURE 10.

ICL3-9 promotes unique conformational changes in the β₂AR that promote G_s coupling. A, monobromobimane is an environmentally sensitive fluorophore that when chemically conjugated to β₂AR-Cys²⁶⁵ can indicate local conformational changes. When the β₂AR is in an inactive state, Cys²⁶⁵-monobromobimane is occupying a hydrophobic pocket and fluorescence is high. Upon receptor activation, a large outward movement of TM6 repositions Cys²⁶⁵ to be solvent-exposed resulting in decreased fluorescence and an increase in λ_max. B, lipid bicelles containing 50 nm monobromobimane-labeled β₂AR were incubated for 10 min at 25 °C with isoproterenol (300 nm or 1 μm) or ICL3-9 (20 or 100 μm) in 20 mm HEPES, pH 7.5, 100 mm NaCl. Fluorescence spectra were gathered by excitation at 370 nm and scanning 430–490 nm at 1.0 nm/s. 0.5% DMSO was included in non-pepducin-stimulated samples. C, lipid bicelles containing 50 nm monobromobimane-labeled β₂AR were incubated for 10 min at 25 °C with 20 μm ICL3-9. 200 nm G_s was then incubated for 20 min at 25 °C in co-treatment studies. 0.1% DMSO was included in non-pepducin-stimulated samples. D, lipid bicelles containing 50 nm monobromobimane-labeled β₂AR were incubated for 10 min at 25 °C with 20 μm ICL3-9A3. 200 nm G_s was then incubated for 20 min at 25 °C in co-treatment studies. 0.1% DMSO was included in non-pepducin-stimulated samples.