Figure 2.

Effect of agonists and partial agonists on fluorescence intensity of FM-β₂AR. (A) The change in intensity of FM-β₂AR in response to the addition of the full agonist ISO and the strong partial agonist epinephrine was reversed by the neutral antagonist ALP. In most experiments, we use the ALP reversal to quantitate the magnitude of the agonist-induced change. We found the ALP reversal to be the most consistent measure for comparison of agonist-induced conformational changes, because ALP reversal occurs over a shorter period relative to agonist responses and therefore is less subject to nonspecific effects on fluorescence intensity (e.g., photobleaching, receptor denaturation) that affect the baseline. ALP alone did not induce any changes in fluorescence, and treatment with ligands did not cause a change in the wavelength of maximum emission (data not shown). (B) Agonist and partial agonist effects on the intensity of FM-β₂AR are compared with an assay of biological efficacy (GTPγS binding). FM-β₂AR was treated with different agonists, and the change in fluorescence was measured at a time equal to 5 times the calculated t_1/2 for each drug. All agonists were used at 100 μM to ensure saturation of the receptors and eliminate the effect of variations in agonist affinities. The ability of these ligands to stimulate GTPγS binding in a β₂AR-Gαs fusion protein was determined, as previously described (41). All data represent experiments performed in triplicate.