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Comparative Study
. 2009 Mar;75(3):534-41.
doi: 10.1124/mol.108.052399. Epub 2008 Dec 23.

Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic receptor activation reveals distinct agonist-specific conformational changes

A Zürn  1 , U Zabel, J-P Vilardaga, H Schindelin, M J Lohse, C Hoffmann
Affiliations
Comparative Study

Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic receptor activation reveals distinct agonist-specific conformational changes

A Zürn et al. Mol Pharmacol. 2009 Mar.

Abstract

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 alpha(2A)-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 approximately 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.

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