EXAMINATION OF LIGAND-INDUCED CONFORMATIONAL-CHANGES IN THE BETA(2) ADRENERGIC-RECEPTOR

The environmentally sensitive and cysteine reactive fluorescent probe, IANBD, was used to monitor ligand-induced structural changes in the b eta 2 adrenergic receptor (beta 2AR) by fluorescent spectroscopy. We f ound that agonists caused a dose-dependent and reversible decrease in fluorescence from the purified IANBD-labeled beta 2AR. This suggested that agonists promote a conformational change in the receptor that lea ds to an increase in the polarity of the environment around one or mor e IANBD labeled cysteines. The wildtype receptor contains eight free c ysteines and mutagenesis and peptide mapping experiments have indicate d that several of these sites are accessible for chemical derivatizati on. Thus, to identify the cysteine(s) involved in the agonist-induced change in fluorescence and thereby map agonist-induced conformational changes in the beta 2AR, we generated a series of mutant receptors hav ing limited numbers of cysteines available for fluorescent labeling. F luorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants showed that IANBD labeled 125Cys and 285Cys are responsible for the observed changes in fluorescence consistent with movements of TM III and VI in response to agonist binding.