Selective interactions between G protein subunits and RGS4 with the C-terminal domains of the mu- and delta-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 Gbetagamma complexes, derived from the heterotrimeric Galphatbeta1gamma1, purified Gbeta1gamma1, or Gbeta 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 Galpha subunit binding. Indeed, while mu-CT was unable to bind any form of Galpha, both the delta-CT and the delta-i3L displayed interactive regions for heterotrimeric Galphatbeta1gamma1, inactive Galpha(GDP) and active Galpha(GTPgammaS). 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 Galpha. 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.