There is considerable evidence for functional interactions between the opioid and adrenergic systems in the spinal cord (Yaksh, 1979
; Roerig et al., 1984
; Wigdor and Wilcox, 1987
; Stone et al., 1997
). Our data suggests that μ
opioid and α2A
adrenergic receptors can physically interact, because the receptor complexes can be isolated from heterologous cells and from neurons by coimmunoprecipitation. This interaction can be enhanced by the addition of selective ligands for either system but not by the addition of both ligands. This is probably caused by conformational changes that render the complexes more stable (with individual agonists) or less stable (with a combination of agonists) to detergents. The alteration of hetero-oligomeric stability by ligands in vitro is an important indication of their ability to induce significant changes in the conformation of the complex, which may be responsible for the observed effects on receptor function. This is supported by our findings with the signaling assays. We find that the presence of α2A
receptors is sufficient to substantially increase the efficacy of morphine in this assay, because this occurs in the absence of α2A
ligands. It is possible that the interaction of the μ
receptor with the inactive α2A
receptor could lead to the stabilization of μ
receptors upon ligand binding to a conformationally active state that efficiently activates G-proteins in response to morphine. Coactivation of α2A
receptors could lead, in turn, to the destabilization of these interactions (supported by our biochemical studies, ), leading to a decrease in the efficacy of G-protein turnover by the two receptors (as seen in the signaling studies, ). Taken together, these results are consistent with the notion that the physical interaction between μ
receptors plays an important role in modulating their signaling and this could be brought about, at least in part, by the agonist-induced changes in receptor conformation and/or association.
The phosphorylation of MAP kinase in response to specific ligands has been extensively used as an assay to test the function of some GPCRs. We find that the presence of α2A
receptors is also sufficient to potentiate the phosphorylation of MAP kinases in response to morphine and that the combination of ligands abrogates this effect. It is also possible that adrenergic receptors, by binding to scaffolding proteins, could bring additional signaling complexes, such as the components of the MAP kinase pathway to the proximity of the μ
receptors, thus enhancing its activity. It is also possible that physical associations lead to changes in the localization of receptors to areas enriched in signaling molecules and that α2A
receptors at least in part, via a physical interaction, help target μ
receptors to “active” areas, such as the lipid rafts. This is not unreasonable, given the case of GABAB
receptors that require associations between the R1 and R2 subunits for proper targeting to the plasma membrane (Margeta-Mitrovic et al., 2000
; Pagano et al., 2001
). For these events to be feasible, the two receptors should be in close proximity, thus influencing their ability to transduce signals. Our results with BRET and colocalization studies support such a notion. Finally, Stone et al. (1997)
showed that in mice harboring mutant α2A
receptors, there was a considerable decrease in the potency of spinally administered morphine, supporting the hypothesis that μ
receptors require functional α2A
receptors for proper functioning in the spinal cord.
Studies with a variety of inflammatory pain models have suggested that the phosphorylation of MAP kinase is important in mediating pain signals (Ciruela et al., 2003
; Dai et al., 2002
). A number of studies have correlated increases in phosphorylated ERK kinases with pain transmission. Our results show that spinal cord neurons are very responsive to opiate or adrenergic stimulation because fairly low doses and short-term treatment lead to a significant increase in the levels of phosphorylated ERK kinases, and these responses are rapidly desensitized. This and the fact that exposure to a combination of ligands lead to a decrease in phosphorylated ERK kinases suggests additional levels of regulation of these receptors in the modulation of analgesia.
In summary, physical interactions between μ and α2A receptors provide a novel mechanism for modulation of receptor function that could have profound effects in the development of analgesia to opioid and adrenergic drugs.