RT Journal Article SR Electronic T1 Fluorescence Resonance Energy Transfer Analysis of α2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 534 OP 541 DO 10.1124/mol.108.052399 VO 75 IS 3 A1 A. Zürn A1 U. Zabel A1 J.-P. Vilardaga A1 H. Schindelin A1 M. J. Lohse A1 C. Hoffmann YR 2009 UL http://molpharm.aspetjournals.org/content/75/3/534.abstract AB Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in α2A-adrenergic receptors in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein and with fluorescein arsenical hairpin binder at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET changes for all three constructs. The strong partial agonists clonidine and dopamine induced partial FRET changes for all constructs. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced FRET-signals were ≈1.5-fold slower than those for norepinephrine in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor. The American Society for Pharmacology and Experimental Therapeutics