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J. Biol. Chem., Vol. 283, Issue 48, 33337-33346, November 28, 2008

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The Third Intracellular Loop Stabilizes the Inactive State of the Neuropeptide Y1 Receptor^*

Melissa J. S. Chee, Karin Mörl^¶, Diana Lindner^¶, Nicole Merten^¶, Gerald W. Zamponi^||, Peter E. Light, Annette G. Beck-Sickinger^¶, and William F. Colmers¹

From the Department of Pharmacology and Centre for Neuroscience, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, the ^¶Institute of Biochemistry, Leipzig University, Brüderstr. 34, 04103 Leipzig, Germany, and the ^||Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Constitutively active G-protein-coupled receptors (GPCRs) can signal even in the absence of ligand binding. Most Class I GPCRs are stabilized in the resting conformation by intramolecular interactions involving transmembrane domain (TM) 3 and TM6, particularly at loci 6.30 and 6.34 of TM6. Signaling by G_i/G_o-coupled receptors such as the Neuropeptide Y1 receptor decreases already low basal metabolite levels. Thus, we examined constitutive activity using a biochemical assay mediated by a G_i/G_q chimeric protein and a more direct electrophysiological assay. Wild-type (WT-Y1) receptors express no measurable, agonist-independent activation, while µ-opioid receptors (MOR) and P2Y₁₂ purinoceptors showed clear evidence of constitutive activation, especially in the electrophysiological assay. Neither point mutations at TM6 (T^6.30A or N^6.34A) nor substitution of the entire TM3 and TM6 regions from the MOR into the Y1 receptor increased basal WT-Y1 activation. By contrast, chimeric substitution of the third intracellular loop (ICL3) generated a constitutively active, Y1-ICL3-MOR chimera. Furthermore, the loss of stabilizing interactions from the native ICL3 enhanced the role of surrounding residues to permit basal receptor activation; because constitutive activity of the Y1-ICL3-MOR chimera was further increased by point mutation at locus 6.34, which did not alter WT-Y1 receptor activity. Our results indicate that the ICL3 stabilizes the Y1 receptor in the inactive state and confers structural properties critical for regulating Y receptor activation and signal transduction. These studies reveal the active participation of the ICL3 in the stabilization and activation of Class I GPCRs.

Received for publication, June 18, 2008 , and in revised form, September 15, 2008.

^* The work was supported by the Canadian Institutes of Health Research (CIHR) Grant MT10520, the German Research Foundation (DFG SFB610, TP A1), and the Saxonian Ministry for Science and Culture (SMWK, Saxonia/Alberta-collaboration 4-7531.50-02-0361-05/1). 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.

¹ To whom correspondence should be addressed: 9-36 Medical Sciences Bldg., Dept. of Pharmacology, University of Alberta, Edmonton, AB T6G 2H7, Canada. Fax: 780-492-4325; E-mail: william.colmers{at}ualberta.ca.

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