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J. Biol. Chem., Vol. 282, Issue 21, 15799-15811, May 25, 2007

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Identification of an Efficacy Switch Region in the Ghrelin Receptor Responsible for Interchange between Agonism and Inverse Agonism^*

Birgitte Holst¹, Jacek Mokrosinski², Manja Lang, Erik Brandt, Rie Nygaard^¶, Thomas M. Frimurer^¶, Annette G. Beck-Sickinger, and Thue W. Schwartz^¶

From the Laboratory for Molecular Pharmacology, The Panum Institute, Blegdamsvej 3, University of Copenhagen, 2200 Copenhagen N, Denmark, Institute of Biochemistry, Faculty of Bioscience, Pharmacy, and Psychology, University of Leipzig, Bruderstrasse 34, 04103 Leipzig, Germany, and ^¶7TM Pharma A/S, Fremtidsvej 3, 2970 Hørsholm, Denmark

The carboxyamidated wFwLL peptide was used as a core ligand to probe the structural basis for agonism versus inverse agonism in the constitutively active ghrelin receptor. In the ligand, an efficacy switch could be built at the N terminus, as exemplified by AwFwLL, which functioned as a high potency agonist, whereas KwFwLL was an equally high potency inverse agonist. The wFw-containing peptides, agonists as well as inverse agonists, were affected by receptor mutations covering the whole main ligand-binding pocket with key interaction sites being an aromatic cluster in transmembrane (TM)-VI and -VII and residues on the opposing face of TM-III. Gain-of-function in respect of either increased agonist or inverse agonist potency or swap between high potency versions of these properties was obtained by substitutions at a number of positions covering a broad area of the binding pocket on TM-III, -IV, and -V. However, in particular, space-generating substitutions at position III:04 shifted the efficacy of the ligands from inverse agonism toward agonism, whereas similar substitutions at position III: 08, one helical turn below, shifted the efficacy from agonism toward inverse agonism. It is suggested that the relative position of the ligand in the binding pocket between this "efficacy shift region" on TM-III and the opposing aromatic cluster on TM-VI and TM-VII leads either to agonism, i.e. in a superficial binding mode, or it leads to inverse agonism, i.e. in a more profound binding mode. This relationship between different binding modes and opposite efficacy is in accordance with the Global Toggle Switch model for 7TM receptor activation.

Received for publication, October 17, 2006 , and in revised form, March 9, 2007.

^* This work was supported in part by grants from the Danish Medical Research Council, The Novo Nordisk Foundation by The Lundbeck Foundation (to B. H.) and by The German Research Foundation Grant SFB 610. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Table S1, and Fig. S1.

² Supported by a Socrates/Erasmus program fellowship.

¹ To whom correspondence should be addressed. Tel.: 45-3532-7602; Fax: 45-3532-7610; E-mail: b.holst{at}molpharm.dk.

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