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| The EMBO Journal
(1997) 16, 6737–6747, doi:10.1093/emboj/16.22.6737 |
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Agonists induce conformational changes in transmembrane domains III and VI of the  2 adrenoceptor |
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| Ulrik Gether1, 4, Sansan Lin1, Pejman Ghanouni1, Juan A. Ballesteros3, Harel Weinstein3 and Brian K. Kobilka1, 2 |
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1 Howard Hughes Medical Institute, Stanford University Medical School, Stanford, CA 94305 USA 2 Division of Cardiovascular Medicine, Stanford University Medical School, Stanford, CA 94305 USA 3 Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029, USA 4 Present address: Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark To whom correspondence should be addressed Ulrik Gether, gether@mfi.ku.dk Brian K. Kobilka, kobilka@cmgm.stanford.edu Received 18 April 1997; Revised 28 July 1997. |
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| Abstract |
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| Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the 2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant 2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors. |
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| Keywords: fluorescence spectroscopy, G protein-coupled receptors, molecular modeling, signal transduction |
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