Selective interactions between G protein subunits and RGS4 with the C-terminal domains of the μ- and δ-opioid receptors regulate opioid receptor signaling

To define receptor subdomains important for protein interaction and identify components of novel signal transduction complexes for the mu- and delta-opioid receptors (mu-OR, delta-OR), we generated glutathione S-transferase fusion proteins of the carboxyl-termini of the mu-opioid receptor (mu-CT), the delta- (delta-CT), and the third intracellular loop of the delta-opioid receptor (delta-i3L) to search for interactive proteins. Results from pull down experiments have demonstrated for the first time that G beta gamma complexes, derived from the heterotrimeric G alpha t beta l gamma l, purified G beta l gamma l, or G beta endogenously present in cell lysates and rat striatal extracts, interact with all mu- and delta-opioid receptor subdomains. On the other hand, the C-terminal peptides of the delta- and the mu-ORs exhibit differential profiles for G alpha subunit binding. Indeed, while mu-CT was unable to bind any form of G alpha, both the delta-CT and the delta-i3L displayed interactive regions for heterotrimeric G alpha t beta l gamma l, inactive G alpha(GDP) and active G alpha(GTP gamma S). 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 mu-CT, the delta-CT as well as delta-i3L in a dose dependent manner. Moreover, RGS4 modulates mu-OR signaling and can form stable heterotrimeric complexes with the activated G alpha. Collectively, our data demonstrate that the C-termini of the mu- and delta-ORs provide direct physical scaffolding in which G protein subunits and RGS4 protein can be bound. (c) 2005 Elsevier Inc. All rights reserved.