Examination of Ligand-Induced Conformational Changes in the β2-Adrenergic Receptor by Fluorescence Spectroscopy

Abstract

Till date, the conformational state of G-protein-coupled receptors has only been inferred indirectly from the activity of the G-protein or the effector enzyme modulated by the G-protein. Fluorescence spectroscopy is used to directly monitor structural changes in the β2-adrenergic receptor. The emission from many fluorescent molecules is strongly dependent on the polarity of the environment in which they are located. Thus, incorporation of fluorescent labels into proteins can be used as sensitive indicators of conformational changes and protein–protein interactions that cause changes in polarity of the environment surrounding the probe. Several β-adrenergic receptor compounds are examined and the effect of these ligands are compared on the fluorescence emission of nitrobenzdioxazol iodoacetamide (IANBD)-labeled β2 receptor with their effect on adenylyl cyclase activity in membranes from SF-9 cells expressing β2 receptors. These studies represent the first direct examination of conformational changes in a G-protein-coupled receptor following ligand binding and the first attempt to correlate the functional properties of different receptor ligands with their effect on the conformational state of a purified, solubilized G-protein-coupled receptor. To gain further insight into the mechanism of receptor activation, another technique is used for directly monitoring receptor structure to investigate a constitutively active receptor. Studies described in this chapter delineate two novel properties of a constitutively activated receptor: structural instability and an exaggerated conformational response to drug binding. These findings, in particular the larger changes in fluorescence observed with constitutively activated mutant (CAM) in response to agonists, are unexpected.