- Opioid Receptor Functional Interaction: Insight Using Receptor-G Protein Fusions

Fusion proteins between a receptor and a pertussis toxininsensitive Gi subunit were used to gain insight into the molecular interactions that take place upon and opioid receptor heterodimerization. When opioid receptor-Gi1 fusions were coexpressed with nonfused opioid receptors in human embryonic kidney 293 cells, or vice versa, receptor heterodimers were detected by coimmunoprecipitation. In pertussis toxin-treated cells, receptor coexpression decreased the amount of guanosine 5 -O-(3-[S]thio)triphosphate ([S]GTP S) incorporated in the fused G protein after the addition of agonists specific for the receptor-Gi1 fusion. In addition, activation of the G protein occurred in heterodimers upon addition of an agonist specific for the nonfused receptor. It remained unaffected by an inverse agonist specific for the receptor-Gi1 fusion. These data suggest that signaling through the receptor-Gi1 fusion protein is impaired in heterodimers and support a mechanism in which activation of the G subunit is promoted by a direct interaction with the nonfused receptor. Alternatively, receptor coexpression did not modify the ligand binding properties for the high-affinity state of the receptor-Gi1 fusion nor the EC50 values for agonist-induced [S]GTP S incorporation in the Gi1 subunit. In addition, no binding competition was observed between and ligands. Together, the data point to opioid receptor heterodimers formed by contact interactions between monomers that retain their structural integrity. Genes coding for , , and opioid receptor types have been identified and isolated from different vertebrates and belong to the G protein-coupled receptor (GPCR) superfamily. The opioid receptors and endogenous opioid peptides form a neuromodulatory system that plays a major role in the control of nociceptive pathways. The opioid system also modulates affective behavior, neuroendocrine physiology, and controls autonomic functions such as respiration, blood pressure, thermoregulation, and gastrointestinal motility. Importantly, the receptors are targets for exogenous narcotic opiate alkaloids that constitute a major class of drugs of abuse (Massotte and Kieffer, 1998; Bodnar and Hadjimarkou, 2003). GPCR signaling is a complex process whose modulation likely proceeds from many intricate factors. Recently, a possible involvement of the receptor oligomerization state has received growing attention since existence of homoand heterodimers has been postulated for an increasing number of GPCRs (for reviews, see Milligan, 2004; Park et al., 2004; Prinster et al., 2005). Arguments in favor of a spatial proximity between receptor molecules mainly arose from coimmunoprecipitation studies and more recently from bioluminescence and fluorescence resonance energy transfer experiments (for review, see Milligan and Bouvier, 2005). However, establishing physical proximity between receptors does not necessarily imply functional interactions between them. Even when modifications of the signaling properties seemed correlated with receptor colocalization, the underlying mechanisms remained largely unknown (Gomes et al.,