Selective interactions between G protein subunits and RGS4 with the C-terminal domains of the A- and y-opioid receptors regulate opioid receptor signaling Zafiroula Georgoussi a , Leonidas Leontiadis a , Georgia Mazarakou a , Manolis Merkouris a , Karren Hyde b , Heidi Hamm b, * a Laboratory of Cellular Signaling and Molecular Pharmacology, Institute of Biology, National Center for Scientific Research ‘‘Demokritos’’ Ag. Paraskevi 15310, Athens, Greece b Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA Received 23 June 2005; accepted 11 July 2005 Available online 24 August 2005 Abstract To define receptor subdomains important for protein interaction and identify components of novel signal transduction complexes for the A- and y-opioid receptors (A-OR, y-OR), we generated glutathione S-transferase fusion proteins of the carboxyl-termini of the A-opioid receptor (A-CT), the y-(y-CT), and the third intracellular loop of the y-opioid receptor (y-i3L) to search for interactive proteins. Results from pull down experiments have demonstrated for the first time that Ghg complexes, derived from the heterotrimeric Gath1g1, purified Gh1g1, or Gh endogenously present in cell lysates and rat striatal extracts, interact with all A- and y-opioid receptor subdomains. On the other hand, the C-terminal peptides of the y- and the A-ORs exhibit differential profiles for Ga subunit binding. Indeed, while A-CT was unable to bind any form of Ga, both the y-CT and the y-i3L displayed interactive regions for heterotrimeric Gath1g1, inactive Ga GDP and active Ga GTPgS . Regulators of G protein signaling (RGS) proteins are another class of proteins that can modulate G protein signaling events. We demonstrate for the first time that RGS4 directly interacts with the A-CT, the y-CT as well as y-i3L in a dose dependent manner. Moreover, RGS4 modulates A-OR signaling and can form stable heterotrimeric complexes with the activated Ga. Collectively, our data demonstrate that the C- termini of the A- and y-ORs provide direct physical scaffolding in which G protein subunits and RGS4 protein can be bound. D 2005 Elsevier Inc. All rights reserved. Keywords: A-, y-opioid receptors; Ga,Ghg subunits; RGS proteins; Signaling complex; GST fusion peptides 1. Introduction All three opioid receptor subtypes (A, y, n) belong to the superfamily of G protein coupled receptors (GPCRs), and mediate the actions of endogenously produced opioid neuropeptides and exogenously administrated opiate drugs. Opioid receptors mediate their responses in the nervous system via coupling to members of the Gi/Go proteins [1] and their activation results in adenylyl cyclase inhibition and/or the regulation of various ion channels and other effector systems [2,3]. Extended observations in respect to opioid receptor signaling mechanisms have demonstrated that the cytoplasmic face of these receptors, and particularly 0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2005.07.003 Abbreviations: GPCRs, G protein coupled receptors; GST, glutathione S-transferase; A-CT, GST fusion of the C-terminal region of the A-opioid receptor; y-CT, GST fusion of the C-terminal region of the y-opioid receptor; y-i3L, GST fusion of the third intracellular loop of the y-opioid receptor; A-OR, A-opioid receptor; y-OR, y-opioid receptor; GTPgS, guanosine 5V-3-O-(thio)triphosphate; GDP, guanosine diphosphate; RGS, regulators of G protein signaling; GAP, GTPase-activating protein; PBS, phosphate-buffered saline; SDS, sodium dodecylsulfate; IPTG, isopro- pylthio-h-galactoside; DAMGO, [d-Ala 2 ,MePhe 4 ,Gly 5 -ol]enkephalin; DTT, dithiothreitol; HA, hemagglutinin; mAChR, muscarinic acetylcholine receptor; i3, intracellular third loop; PLC, phospholipase C; HEK293, human embryonic kidney. * Corresponding author. E-mail address: heidi.hamm@vanderbilt.edu (H. Hamm). Cellular Signalling 18 (2006) 771 – 782 www.elsevier.com/locate/cellsig