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 μ- and δ-opioid receptors (μ-OR, δ-OR), we generated glutathione S-transferase fusion proteins of the carboxyl-termini of the μ-opioid receptor (μ-CT), the δ- (δ-CT), and the third intracellular loop of the δ-opioid receptor (δ-i3L) to search for interactive proteins. Results from pull down experiments have demonstrated for the first time that Gβγ complexes, derived from the heterotrimeric Gαtβ1γ1, purified Gβ1γ1, or Gβ endogenously present in cell lysates and rat striatal extracts, interact with all μ- and δ-opioid receptor subdomains. On the other hand, the C-terminal peptides of the δ- and the μ-ORs exhibit differential profiles for Gα subunit binding. Indeed, while μ-CT was unable to bind any form of Gα, both the δ-CT and the δ-i3L displayed interactive regions for heterotrimeric Gαtβ1γ1, inactive GαGDP and active GαGTPγS. 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 μ-CT, the δ-CT as well as δ-i3L in a dose dependent manner. Moreover, RGS4 modulates μ-OR signaling and can form stable heterotrimeric complexes with the activated Gα. Collectively, our data demonstrate that the C-termini of the μ- and δ-ORs provide direct physical scaffolding in which G protein subunits and RGS4 protein can be bound.