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 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.
Footnotes
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This work was supported by the Deutsche Forschungsgemeinschaft [Grant SFB487] “Regulatory Membrane Proteins”; by the Ernst Jung Award for Medicine; and by the Fonds der Chemischen Industrie.
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ABBREVIATIONS: GPCR, G-protein-coupled receptor; CFP, cyan fluorescent protein; CL, clonidine; DA, dopamine; EDT, 1,2-ethane dithiol; FlAsH, 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein; FRET, fluorescence resonance energy transfer; HBSS, Hanks' balanced salt solution; NE, norepinephrine; NF, norphenephrine; OC, octopamine; TM, transmembrane domain; [3H]RX821002, 3H-2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; HEK, human embryonic kidney; PDB, Protein Data Bank; [35S]GTPγS, guanosine 5′-O-(3-[35S]thio)triphosphate.
- Received October 2, 2008.
- Accepted December 22, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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