We have previously reported that δ-receptors exist as homodimers and that agonist treatment modulates the level of dimers12
. Using western blotting, we examined lysates from cells expressing κ-receptors tagged with a Flag epitope to see if κ-receptors exist as dimers. We found that most κ-receptors exist as dimers of relative molecular mass (Mr
) 130,000, regardless of the presence or absence of a crosslinker (). The dimers are stable in 10% SDS buffer (not shown); this is unexpected, because δ-receptors show little or no dimeric forms in the absence of the crosslinker; crosslinking is required to stabilize δ-receptor dimers (). The identity of this dimer was confirmed by immunoprecipitation experiments using differentially tagged receptors. We found that an antibody to the myc
-tagged receptor can co-precipitate Flag-tagged receptors from cells expressing both myc
-tagged and Flag-tagged receptors (). The dimerization of κ-receptors is not induced by detergents or extraction conditions, as the receptors could be co-precipitated under a variety of conditions, but only from cells co-expressing myc
-and Flag-tagged receptors (), and not from a mixture of cells individually expressing the receptors (). κ-dimers are destabilized in the presence of reducing agents (), suggesting the involvement of disulphide bonds in receptor dimerization; recently, both the metabotropic glutamate receptor 5 and the calcium-sensing receptor have been shown to dimerize through disulphide bonds13,14
. We found that agonist treatment does not induce monomerization of κ-receptor dimers (), in contrast to δ-receptor dimers, which monomerize in the presence of agonists12
. These results indicate that the properties of κ-receptor dimers and δ-receptor dimers may be different.
Figure 1 Characteristics of κ-opioid-receptor homodimers. a, Immunoblotting of lysates from cells expressing Flag–κ receptors or Flag–δ receptors. b, c, Myc-tagged κ-receptors can be co-precipitated only from cells (more ...)
We examined the ability of κ-receptors to heterodimerize with δ-or µ-receptors by co-expressing myc-tagged κ-receptors with either Flag-tagged δ-receptors or Flag-tagged µ-receptors. Flag-tagged δ-receptors were detected in material immunoprecipitated using antibodies specific for myc-tagged κ-receptors (). In contrast, Flag-tagged µ-receptors could not be detected under similar co-precipitation conditions (). These results indicate that κ-receptors selectively dimerize with δ- but not with µ-opioid receptors. κ–δ heterodimers are stable in a variety of detergents and are not induced during solubilization/immunoprecipitation procedures (). They are also destabilized by a reducing agent (), suggesting a role for disulphide bonds inκ–δ heterodimerization.
Figure 2 Characterization of κ–δ heterodimers. a, κ–δ heterodimers can be immunoprecipitated only from myc–κ- and Flag-δ-expressing cells and not from myc–κ- and Flag–µ-expressing (more ...)
We next examined the effect of heterodimerization on receptor trafficking using cells co-expressing κ- and δ-receptors. Etorphine is a potent, non-selective opioid agonist that binds both δ- and κ-receptors with high affinity. As was shown previously15–17
, etorphine can induce robust internalization of δ- but not κ-receptors in cells individually expressing these receptors (). In contrast, etorphine cannot induce substantial internalization of δ-receptors in cells expressing both κ- and δ-receptors; the internalization of δ-homodimers in these cells could account for the observed ~25–30% reduction in surface fluorescence (). These results suggest a role for heterodimerization in altering the trafficking properties of these receptors.
We compared the ligand-binding properties of κ–δ heterodimers with those of κ- or δ-receptors (), and examined the ability of highly selective agonists18,19
to compete with 3
H-diprenorphine (a non-selective opioid antagonist) in membranes from cells expressing either κ- or δ- or both κ- and δ-receptors. (). We found that κ-receptors have high affinities for the κ-selective agonist (U69593) and antagonist (norbinaltorphimine). Similarly, δ-receptors have high affinities for the δ-selective agonist ([d
]enkephalin; DPDPE) and antagonist (TIPPΨ; ref. 21
). In contrast, κ–δ heterodimers show no significant affinity for either κ- or δ-selective agonists or antagonists (; ). However, the heterodimer shows a strong affinity for partially selective ligands (). The properties of these heterodimers are virtually identical to those of the previously reported κ-2-receptor subtype22
. Although several studies have reported the presence of other subtypes of κ- (ref. 23
) and δ- (refs 24, 25
) opioid receptors, complementary DNAs corresponding to these subtypes have not been identified despite large-scale efforts by several laboratories. Recent work with δ-receptor knockout mice shows that both the δ1 and δ2 receptor subtypes are eliminated in these animals, indicating that the δ-receptor locus may encode both of these subtypes. It is possible that the heterodimerization of δ- or κ-receptors with other GPCRs could form a molecular basis for other receptor subtypes.
Ligand-binding properties of κ–δ heterodimer
Figure 3 Ligand binding and functional properties. a–c, Competition of 3H-diprenorphine binding by U69593 (square), norbinaltorphimine (triangle), diprenorphine (star), DPDPE (circle) and TIPPΨ (diamond) in membranes from cells expressing κ- (more ...)
We next examined whether the κ–δ heterodimer binds selective agonists synergistically. In the presence of a δ-selective agonist (DPDPE), a κ-agonist (U69593) binds the heterodimer with high affinity (, ). Similarly, in the presence of the κ-selective agonist (U69593), the δ-agonist (DPDPE) binds with high affinity (, ). Interestingly, whereas a combination of two selective antagonists also binds with high affinity, a combination of a selective agonist (U69593) and a selective antagonist (TIPPΨ) does not (). Also, synergistic binding is not observed in membranes from cells individually expressing κ- or δ-receptors (not shown). Taken together, these results imply that κ–δ heterodimerization results in a new binding site that is able to bind highly selective ligands synergistically.
We examined whether the synergistic binding of agonists leads to increased effector function. The activation of opioid receptors by agonists results in decreased levels of intracellular cyclic AMP and increased levels of phosphorylated mitogen-activated protein kinase (MAPK)26
. We found that the potency of individual agonists in reducing intracellular cAMP levels is ~10–20-fold lower than that of the two combined (). Similarly, we found a significant potentiation of MAPK phosphorylation by simultaneous treatment of cells with both agonists as compared to individual agonists (). These results strongly suggest that the κ–δ heterodimer represents a functional receptor that is activated synergistically by selective ligands.
Our data provide biochemical and functional evidence for opioid-receptor heterodimerization. This is the first direct evidence for the heterodimerization of opioid-receptor types and for two fully functional GPCRs. The heterodimers have greatly reduced affinities for their selective ligands. Interestingly, selective agonists can cooperatively bind to heterodimers and induce synergistic functional responses. Heterodimerization could be a mechanism for activating the receptors on the co-release of selective endogenous peptides. Alternatively, κ–δ-opioid-receptor heterodimers could represent a hitherto uncharacterized receptor for a specific endogenous opioid peptide. The number of endogenous opioid peptides is far greater than the number of cloned opioid receptors27
. Opioid-receptor subtypes resulting from heterodimerization of opioid receptors with other GPCRs could be targets for the action of these endogenous peptides. The physical interactions between GPCRs has enormous ramifications for our understanding of how their actions are regulated. Heterodimerization of opioid receptors also points to additional targets for the development of therapeutic drugs.