LIGAND STABILIZATION OF THE BETA(2) ADRENERGIC-RECEPTOR - EFFECT OF DTT ON RECEPTOR CONFORMATION MONITORED BY CIRCULAR-DICHROISM AND FLUORESCENCE SPECTROSCOPY

Treatment of the beta(2) adrenergic receptor with the reducing agent d ithiothreitol (DTT) is known to abolish ligand binding to the receptor . Interestingly, the loss of binding can be prevented by preoccupation of the receptor with ligand, It is unclear, however, whether the liga nd blocks access of DTT to the receptor, or the ligand stabilizes the receptor structure. In the present study, we have utilized circular di chroism (CD) and intrinsic tryptophan fluorescence to directly probe s tructural changes in the beta(2) adrenergic receptor in response to DT T treatment. Analysis of CD spectra of purified beta(2) receptor in th e detergent micelle indicated that the receptor has an alpha-helix con tent of 60%, which is substantially more than what would be attributed to the seven transmembrane domains. The alpha-helix content was uncha nged in the presence of DTT, suggesting that DTT treatment does not al ter the secondary structure of the receptor. In contrast, the tryptoph an fluorescence spectra demonstrated that DTT induces a reversible con formational change of the beta(2) receptor. Thus, DTT caused a red-shi ft in the maximum emission wavelength of the intrinsic tryptophan fluo rescence. The change in emission spectrum correlated with a loss in th e ability of the receptor to bind antagonist. Both changes in receptor binding and fluorescence emission were reversible, as removal of DTT allowed the receptor to restore 70% of ligand binding and return to th e initial emission spectrum. Furthermore, we found adrenergic antagoni sts were able to slow the rate of the conformational change induced by DTT but not the rate of disulfide reduction, suggesting that the anta gonists stabilize the structure of the reduced receptor.