Ligand Stabilization of the β2 Adrenergic Receptor: Effect of DTT on Receptor Conformation Monitored by Circular Dichroism and Fluorescence Spectroscopy Export

@article{citeulike:1697894, abstract = {Abstract: Treatment of the 2 adrenergic receptor with the reducing agent dithiothreitol (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 ligand blocks access of DTT to the receptor, or the ligand stabilizes the receptor structure. In the present study, we have utilized circular dichroism (CD) and intrinsic tryptophan fluorescence to directly probe structural changes in the 2 adrenergic receptor in response to DTT treatment. Analysis of CD spectra of purified 2 receptor in the detergent micelle indicated that the receptor has an -helix content of 60\%, which is substantially more than what would be attributed to the seven transmembrane domains. The -helix content was unchanged in the presence of DTT, suggesting that DTT treatment does not alter the secondary structure of the receptor. In contrast, the tryptophan fluorescence spectra demonstrated that DTT induces a reversible conformational change of the 2 receptor. Thus, DTT caused a red-shift in the maximum emission wavelength of the intrinsic tryptophan fluorescence. The change in emission spectrum correlated with a loss in the 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 the initial emission spectrum. Furthermore, we found adrenergic antagonists were able to slow the rate of the conformational change induced by DTT but not the rate of disulfide reduction, suggesting that the antagonists stabilize the structure of the reduced receptor.}, address = {Howard Hughes Medical Institute, Division of Cardiovascular Medicine, Stanford University Medical School, Stanford, California 94305}, author = {Lin, S. and Gether, U. and Kobilka, B. K.}, citeulike-article-id = {1697894}, citeulike-linkout-0 = {http://dx.doi.org/10.1021/bi961619+}, citeulike-linkout-1 = {http://pubs.acs.org/doi/abs/10.1021/bi961619+}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/8931540}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=8931540}, day = {19}, doi = {10.1021/bi961619+}, journal = {Biochemistry}, keywords = {gpcr, imp, ligand}, month = {November}, number = {46}, pages = {14445--14451}, posted-at = {2008-05-27 17:25:29}, priority = {2}, title = {Ligand Stabilization of the \&\#x03B2;2 Adrenergic Receptor: Effect of DTT on Receptor Conformation Monitored by Circular Dichroism and Fluorescence Spectroscopy}, url = {http://dx.doi.org/10.1021/bi961619+}, volume = {35}, year = {1996} }

TY - JOUR ID - citeulike:1697894 L3 - citeulike-article-id:1697894 N2 - Abstract: Treatment of the 2 adrenergic receptor with the reducing agent dithiothreitol (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 ligand blocks access of DTT to the receptor, or the ligand stabilizes the receptor structure. In the present study, we have utilized circular dichroism (CD) and intrinsic tryptophan fluorescence to directly probe structural changes in the 2 adrenergic receptor in response to DTT treatment. Analysis of CD spectra of purified 2 receptor in the detergent micelle indicated that the receptor has an -helix content of 60%, which is substantially more than what would be attributed to the seven transmembrane domains. The -helix content was unchanged in the presence of DTT, suggesting that DTT treatment does not alter the secondary structure of the receptor. In contrast, the tryptophan fluorescence spectra demonstrated that DTT induces a reversible conformational change of the 2 receptor. Thus, DTT caused a red-shift in the maximum emission wavelength of the intrinsic tryptophan fluorescence. The change in emission spectrum correlated with a loss in the 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 the initial emission spectrum. Furthermore, we found adrenergic antagonists were able to slow the rate of the conformational change induced by DTT but not the rate of disulfide reduction, suggesting that the antagonists stabilize the structure of the reduced receptor. IS - 46 JF - Biochemistry AD - Howard Hughes Medical Institute, Division of Cardiovascular Medicine, Stanford University Medical School, Stanford, California 94305 EP - 14451 TI - Ligand Stabilization of the β2 Adrenergic Receptor: Effect of DTT on Receptor Conformation Monitored by Circular Dichroism and Fluorescence Spectroscopy VL - 35 SP - 14445 KW - gpcr KW - imp KW - ligand AU - Lin, S AU - Gether, U AU - Kobilka, BK PY - 1996/11/19/ UR - http://dx.doi.org/10.1021/bi961619+ ER -