G-protein-coupled receptor heterodimerization modulates receptor function

Abstract

The opioid system modulates several physiological processes, including analgesia, the stress response, the immune response and neuroendocrine function. Pharmacological and molecular cloning studies have identified three opioid-receptor types, delta, kappa and mu, that mediate these diverse effects. Little is known about the ability of the receptors to interact to form new functional structures, the simplest of which would be a dimer. Structural and biochemical studies show that other G-protein-coupled receptors (GPCRs) interact to form homodimers. Moreover, two non-functional receptors heterodimerize to form a functional receptor, suggesting that dimerization is crucial for receptor function. However, heterodimerization between two fully functional receptors has not been documented. Here we provide biochemical and pharmacological evidence for the heterodimerization of two fully functional opioid receptors, kappa and delta. This results in a new receptor that exhibits ligand binding and functional properties that are distinct from those of either receptor. Furthermore, the kappa-delta heterodimer synergistically binds highly selective agonists and potentiates signal transduction. Thus, heterodimerization of these GPCRs represents a novel mechanism that modulates their function.

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 expressing both myc and Flag-tagged receptors (b) under a variety of extraction conditions and not from a mixture of cells individually expressing these receptors (c). Expression of myc- or Flag-tagged receptors was confirmed by immunoblotting with the appropriate antisera (right panel). d, e, Treatment of cells expressing κ-receptors with 1 mM DTT for 30 min followed by 5 mM iodacetamide (IAM) or N-ethylmaleimide (NEM) results in monomerization (d), whereas treatment with 100 nM agonists for 60 min does not (e). Immunoblotting experiments used anti-Flag antibodies; immunoprecipitation experiments used anti-myc antibodies.

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 cells. b, κ–δ heterodimers can be immunoprecipitated under a variety of extraction conditions and not from a mixture of cells individually expressing these receptors. c, Expression of myc- or Flag-tagged receptors in each cell line was confirmed by immunoblotting with the appropriate antisera (right panel). Treatment with β-mercaptoethanol 5% (β–ME) for 5 min results in the destabilization of dimers. d, Internalization of receptors in response to 1 µM etorphine for 60 min. Stippled bars, myc–δ; shaded bars, Flag–κ. Significant differences from untreated controls are indicated; *P < 0.05; ***P < 0.005 (n = 3). Immunoblotting experiments used anti-Flag antibodies; immunoprecipitation experiments used anti-myc antibodies.

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- (a), δ- (b) or κ- and δ- (c) receptors. d, Displacement of ³H-diprenorphine by U69593 in the presence of 10 µM DPDPE (triangle) or DPDPE in the presence of 10 µM U69593 (diamond). e, f, Decrease in intracellular cAMP (e) or increase in phospho-MAPK (f) by U69593 (square), DPDPE (circle) or U69593 + DPDPE (triangle). In e, the 50% inhibitory concentrations (nM) were: U69593, 1.3 ± 0.7; DPDPE, 0.9 ± 0.4; U69593 + DPDPE, 0.06 ± 0.03. Activation of homodimers in these cells could account for the effect seen by individual agonists. Error bars represent s.e.m. (n = 3–4).