Short communication Mutation S363A in the human y-opioid receptor selectively reduces down-regulation by a peptide agonist Edita Navratilova a , Eva V. Varga a,e , Dagmar Stropova a , Janelle C. Jambrosic a , William R. Roeske a,d,e , Henry I. Yamamura a,b,c,d,e , * a Department of Pharmacology, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA b Department of Biochemistry, The University of Arizona, Tucson, AZ 85724, USA c Department of Psychiatry, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA d Department of Medicine, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA e The Sarver Heart Center, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA Received 25 November 2003; accepted 28 November 2003 Abstract Chemically distinct opioid agonists have different abilities to down-regulate opioid receptors. The present study investigated the role of Ser 363 in human y-opioid receptor down-regulation by a y-selective peptide- and non-peptide agonist. Cyclic[D-Pen 2 ,D-Pen 5 ]enkephalin (DPDPE)-mediated down-regulation was significantly attenuated by a S363A mutation. In contrast, this mutation had no effect on down- regulation by (+)-4-[(aR)-a-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide (SNC80). These results demonstrate that the molecular mechanism of the human y-opioid receptor down-regulation is agonist-specific. D 2003 Elsevier B.V. All rights reserved. Keywords: y-Opioid receptor; Trafficking, agonist-directed; Down-regulation Human y-opioid receptor down-regulation is thought to be involved in the development of analgesic drug tolerance (Fleming and Taylor, 1995). Agonist-activation of G-pro- tein-coupled receptors is typically followed by receptor phosphorylation and recruitment of h-arrestins, leading to the termination of G-protein signaling. In addition, h- arrestin binding facilitates receptor endocytosis via a cla- thrin- and dynamin-dependent mechanism. The internalized receptors are subsequently trafficked into lysosomes for degradation (down-regulation), or to recycling vesicles for re-incorporation into the plasma membrane. Consequently, the intracellular trafficking of the receptor participates in the regulation of receptor signaling. It has been demonstrated that the fate of the internalized G- protein-coupled receptors is both receptor- and cell type- specific (Tsao and von Zastrow, 2000). We have previously found that the trafficking of the human y-opioid receptor is also agonist-specific. In Chinese hamster ovary (CHO) cells stably expressing a human y-opioid receptor, the non-peptide agonist (+)-4-[(aR)-a-((2S,5R)-4-allyl-2,5-dimethyl-1-piper- azinyl)-3-methoxybenzyl]N,N-diethylbenzamide (SNC80) down-regulates the receptor more efficiently than the peptide agonist cyclic[D-Pen 2 ,D-Pen 5 ]enkephalin (DPDPE) (Okura et al., 2003). Furthermore, truncation of the carboxyl-terminus at Gly 338 completely abolishes DPDPE-, but not SNC80- mediated down-regulation (Okura et al., 2000). These results led to the hypothesis that structurally distinct agonists utilize specific receptor domains to regulate receptor trafficking; namely, DPDPE, but not SNC80, requires an intact carboxyl- terminus to down-regulate the human y-opioid receptor. In the present study, we investigated the role of Ser 363 , the primary phosphorylation site in the distal C-terminal domain of the y-opioid receptor (Kouhen et al., 2000), in agonist- directed trafficking. The human y-opioid receptor was subcloned in pcDNA3 expression vector. A mutation (Ser 363 to Ala) was intro- duced using the Quick Change site-directed mutagenesis method (Stratagene). The presence of the Ser 363 to Ala mutation was verified by nucleotide sequencing at the University of Arizona sequencing facility. A stable cell line expressing the human y-opioid receptor (S363A) mutant 0014-2999/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2003.11.067 * Corresponding author. Department of Pharmacology, College of Medicine, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA. Tel.: +1-520-626-7381; fax: +1-520-626-2204. E-mail address: hiy@u.arizona.edu (H.I. Yamamura). www.elsevier.com/locate/ejphar European Journal of Pharmacology 485 (2004) 341 – 343