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J. Biol. Chem., Vol. 279, Issue 51, 53806-53817, December 17, 2004

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Common Structural Basis for Constitutive Activity of the Ghrelin Receptor Family^*

Birgitte Holst, Nicholas D. Holliday¶, Anders Bach, Christian E. Elling||, Helen M. Cox¶, and Thue W. Schwartz||

From the Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200, Copenhagen, Denmark, ^¶Centre for Neuroscience Research, King's College London, Guy's Campus, London, SE1 1UL, United Kingdom, and ^||7TM Pharma A/S, Fremtidsvej 3, DK-2970, Hørsholm, Denmark

Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G_q, phospholipase C pathway was 50% of their maximal capacity as determined through inositol phosphate accumulation. These two receptors also showed very high constitutive activity in activation of cAMP response element-driven transcription. GPR39 displayed a clear but lower degree of constitutive activity through the inositol phosphate and cAMP response element pathways. In contrast, GPR39 signaled with the highest constitutive activity in respect of activation of serum response element-dependent transcription, in part, possibly, through G_12/13 and Rho kinase. Antibody feeding experiments demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically be tuned up and down depending on the size and hydrophobicity of the side chain in position VI:16 in the context of an aromatic residue at VII:09 and a large hydrophobic residue at VII:06. It is concluded that the three ghrelin-like receptors display an unusually high degree of constitutive activity, the structural basis for which is determined by an aromatic cluster on the inner face of the extracellular ends of TMs VI and VII.

Received for publication, July 8, 2004 , and in revised form, September 14, 2004.

^* This study was supported in part by grants from The Novo Nordisk Foundation and the Danish Medical Research Council (to B. H.), a 7TM Biotech Competence Center grant from the Danish Medical Research Council (to T. W. S.), and by the Kimmel Cancer Foundation (to H. M. C.). 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.

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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