Expression of the Third Intracellular Loop of the -Opioid Receptor Inhibits Signaling by Opioid Receptors and Other G Protein-Coupled Receptors

To explore the feasibility of developing inhibitors of signaling by opioid receptors and other G protein-coupled receptors (GPCRs) that use the same G protein pool, we investigated the capacity of a minigene encoding the third intracellular loop of the -opioid receptor ( -i3L) to act as competitive antagonist of the receptor-G protein interface interaction. In -i3L-expressing cells, the peptide blocked high-affinity agonist binding to both the and the -opioid ( -OR and -OR) and attenuated opioid and 2-adrenergic receptor ( 2AR)-dependent [ S]guanosine5 -O-(3-thio)triphosphate binding. Furthermore, -i3L expression resulted in inhibition of -, -OR-, and 2AR-receptormediated cAMP accumulation, whereas the cAMP response produced by activation of the 2-adrenergic receptor was unaffected, suggesting that the inhibitory effects of -i3L expression were selective for Gi/Go proteins. Moreover, although -i3L expression also attenuated drastically phospholipase C accumulation and Ca release following and -OR stimulation, it failed to inhibit carbachol-mediated stimulation of inositol phosphate accumulation in M1-muscarinic receptor-expressing human embryonic kidney 293 cells. Finally, we also examined the effects of -i3L expression on the regulation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway. Our results demonstrate that, although ERK activation by and -ORs is attenuated by the presence of -i3L, ERK activation mediated by 2AR remained unaffected. Collectively, our data demonstrate that the -i3L can be used as potent inhibitor of G protein signaling for various GPCRs that use a common pool of G proteins. Opiate drugs mediate their analgesic, euphoriant, and rewarding effects by activating opioid receptors (Waldhoer et al., 2004). Pharmacological and molecular studies have demonstrated the existence of three opioid receptor subtypes, , , and ( -OR, -OR, and -OR), that couple to Gi/Go types of G proteins to inhibit adenylyl cyclase (Law et al., 1999, 2000). Opioid receptors regulate a number of second messenger systems, such as phospholipase C (PLC), mitogen-activated kinase (MAPK), and various ion channels and other signaling intermediates (Standifer and Pasternak, 1997; Law et al., 2000; Lo and Wong, 2004; Mazarakou and Georgoussi, 2005). Such diverse signaling events are mediated by various G protein subtypes in a PTX-sensitive or -insensitive manner depending on the system studied and the opioid agonists used (Georgoussi et al., 1993; Chan et al., 1995; Garzon et al., 1997; SanchezBlazquez et al., 2001; Lo and Wong, 2004). Opioid receptors are activated by both endogenously produced opioid peptides and exogenously administered opiate compounds, some of which are not only among the most effective analgesics known but also highly addictive drugs of abuse. Various agents that act as agonists or antagonists of GPCRs represent the most common type of drug in clinical use today. Irrespective of chemical composition, these agents share a common feature of acting extracellularly and either mimicking or precluding agonist binding to the receptor. This work was supported by grants from the Hellenic General Secretariat of Science and Technology, Greek Ministry of Development (97EKBAN2-112, EPETII) and the pharmaceutical company ELPEN Pharmaceutical Co. Inc. (Attica, Greece). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.089946. ABBREVIATIONS: R, -opioid receptor; R, -opioid receptor; PLC, phospholipase C; MAPK, mitogen-activated kinase; PTX, Bordetella pertussis toxin; GPCR, G protein-coupled receptor; i3L, third intracellular loop; 2AR, 2-adrenergic receptor; 2AR, 2-adrenergic receptor; DADLE, [D-Ala,D-Leu]-enkephalin; DAMGO, [D-Ala,N-MePhe,Gly-ol]-enkephalin; GppNHp, guanosine 5 -[ , -imido]triphosphate; GST, glutathione S-transferase; PCR, polymerase chain reaction; HEK, human embryonic kidney; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DSLET, [D-Ser,Leu]-enkephalin-Thr; GTP S, [S]guanosine 5 -O-(3-thio)triphosphate; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; EGF, epidermal growth factor; ERK, extracellular signalregulated kinase; UK-14,304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine. 0022-3565/05/3153-1368–1379$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 315, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 89946/3064687 JPET 315:1368–1379, 2005 Printed in U.S.A. 1368 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from Thus, targeting the external ligand binding sites of GPCRs is the generally accepted strategy for designing antagonists. An alternative approach for achieving functional inhibition of GPCRs is the targeting of the receptor-G protein interface with agents that block the intracellular coupling of the receptors to the G protein. Such an approach differs fundamentally from classical GPCR pharmacology, because the blockage of receptor-G protein coupling may result in the achievement of G protein rather than receptor-specific antagonism. Several successful applications of this strategy, using polypeptides derived from the putative contact surfaces of the receptor or the G subunits of G proteins, have been reported (Luttrell et al., 1993; Hawes et al., 1994; Gilchrist et al., 1999; Vanhauwe et al., 2002). For example, expression of peptides derived from the third intracellular domains of the Gq/11-coupled 1B-adrenergic and M1-muscarinic acetylcholine receptors, the Gi-coupled 2A-adrenergic and M2-acetylcholine receptors, and the Gs-coupled D1A dopamine receptor have been shown to inhibit G protein mediated-receptor signaling (Luttrell et al., 1993; Hawes et al., 1994). On the other hand, minigene plasmids encoding oligopeptides representing the carboxyl termini of various G proteins, such as Gi , Gq , Gs , and G 12,13, have also been used to determine the contribution of different G protein pools to the signaling of various GPCRs (Gilchrist et al., 1999, 2001; Feldman et al., 2002). Studies related to opioid receptor signaling mechanisms have demonstrated that the cytoplasmic face of these receptors, particularly the third intracellular loop and the COOH tail, are critical in mediating signal transduction by G proteins. Indeed, previous observations using receptor-derived peptides from specific regions of the and the -ORs have shown that the third intracellular loop (i3L) and the juxtamembranous region of the C-terminal tail of the and -OR are critical for functional receptor-G protein interaction (Merkouris et al., 1996; Georgoussi et al., 1997). These observations have confirmed that there are different determinants for receptor-G protein coupling and G protein activation. Given these observations and the importance of the i3 loop for receptor-G protein coupling and G protein-mediated signal transduction upon receptor activation, we have investigated the feasibility of developing an analog that could inhibit G protein-mediated downstream effects for activated GPCRs that couple to a common G protein pool. Accordingly, we developed a minigene construct capable of directing the expression of the i3 loop of the murine -OR in various cells lines and measured its ability to inhibit the downstream signaling of a number of receptors that couple to Gi/Go proteins. Our results suggest that the presence of the -i3L minigene construct in the various cell lines not only prevents G protein coupling of -OR and -OR but also affects the functional coupling of other GPCRs that selectively interact with the same Gi/Go protein population. In contrast, -i3L expression has no effect on Gsor Gq-coupled receptors. These results provide insight into the importance of the interplay between GPCRs and selective G protein pools in cellular regulation, suggest that opioid receptor-derived peptides can be used as selective inhibitors for such interactions, and point to novel strategies related to drug development for physiological perturbations caused by excessive GPCR activity. Materials and Methods Materials. [H]DADLE (57 Ci/mmol), [H]DAMGO (60 Ci/mmol), [H]diprenorphine (50 Ci/mmol), and [H]adenine (23 Ci/mmol) were from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All receptor ligands were from Sigma-Aldrich (St. Louis, MO). The rat myc-tagged -OR (in the pcDNA3 vector) was generously provided by Dr. S. George (University of Toronto, Toronto, ON, Canada). Lipofectamine was purchased from Invitrogen (Carlsbad, CA). Protein A and protein AG-Sepharose beads were from GE Healthcare. PTX, GppNHp, protease, phosphatase inhibitor cocktails, and all other reagents were purchased from Sigma-Aldrich. Construction of the -i3L and GST-i3L Minigene Encoding the Third Intracellular Loop of the -OR. Two partially complementary oligonucleotides encoding the 23 amino acids of the third intracellular loop of murine -OR (amino acids 239–261) were engineered into 5 and 3 ends. The 5 end contained a BamHI site followed by the ribosome binding consensus sequence (5 -GCC GCC ACC-3 ), a methionine (ATG) for translation initiation, and a glycine (GGA) to protect the ribosome-binding site during translation and the nascent peptide from proteolytic degradation. An EcoRI site was designed at the 3 end immediately following the translation stop codon (TAA). Partially complementary oligonucleotides were allowed to anneal, and full-length (completely complementary) doublestranded DNA was synthesized using T4 DNA polymerase in the presence of dNTPs. After digestion with the restriction enzymes BamHI and EcoRI, the cDNA was ligated to the eukaryotic plasmid vector pcDNA3. The presence of the insert was verified by automated dideoxynucleotide sequencing (ABI Prism 377 DNA sequencer; Applied Biosystems, Foster City, CA). The GST fusion construct encompassing the i3L (amino acids 239–261) of the -OR was generated using the cDNA clone of the rat -OR, as template for PCR and the TOPO cloning procedure (Invitrogen). Transformants were transferred from entry clones to the destination vector pDest-27 (for N-terminal GST fusion protein expression) using the GATEWAY cloning technology (Invitrogen). Cell Culture and Transfection. HEK293 cells, stably expressing the EYMPME (EE)-tagged -opioid, -opioid, and 2-adrenergic receptors, were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin under 5% CO2 at 37°C. Rat-1 fibroblasts stably expressing the 2-adrenergic receptor (generously provided by Prof. G. Milligan, University of Glasgow, Glasgow, Scotland) were grown in the same medium. Transfections were performed on 80% confluent monolayers in 100-mm dishes using Lipofectamine according to the manufacturer’s instructions (Invitrogen). All assays on transiently transfected cells were performed after