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J. Biol. Chem., Vol. 284, Issue 2, 1324-1336, January 9, 2009

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The C-terminal Tail of CRTH2 Is a Key Molecular Determinant That Constrains G_i and Downstream Signaling Cascade Activation^*

Ralf Schröder, Nicole Merten, Jesper Mosolff Mathiesen, Lene Martini^¶, Anamarija Kruljac-Letunic^||, Friederike Krop^||, Andree Blaukat^||, Ye Fang^**, Elizabeth Tran^**, Trond Ulven, Christel Drewke, Jennifer Whistler^¶, Leonardo Pardo, Jesús Gomeza, and Evi Kostenis¹

From the Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany, the Department of Molecular Pharmacology, Zealand Pharma A/S, Smedeland 26B, DK-2600 Glostrup, Denmark, the ^¶Ernest Gallo Clinic and Research Center, University of California, San Francisco, Emeryville, California 94608, ^||TA Oncology, Merck Serono Research, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany, ^**Biochemical Technologies, Science and Technology Division, Corning Inc., Corning, New York 14831, the Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark, the Laboratorio de Medicina Computacional, Unitat de Bioestadistica, Facultat de Medicina, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain

Prostaglandin D₂ activation of the seven-transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases, such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, the presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced G_i and ERK1/2 activation despite enhanced constitutive and agonist-mediated internalization. Enhanced activation of G_i proteins and downstream signaling cascades is probably due to the inability of the tail-truncated receptor to recruit β-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits G_i signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.

Received for publication, September 4, 2008 , and in revised form, October 26, 2008.

^* 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 Figs. 1 and 2.

¹ To whom correspondence should be addressed: Institute for Pharmaceutical Biology, Dept. of Molecular, Cellular, and Pharmacobiology, University of Bonn, Nussallee 6, 53115 Bonn, Germany. Tel.: 49-228-732678/733194; Fax: 49-228-733250; E-mail: kostenis{at}uni-bonn.de.

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