Agonist receptor ligands often down-regulate their receptor targets by internalization into the cell, leading to desensitization of the signaling mechanism. This can represent a problem for use of such drugs in long-term treatment. In the use of opioid agonists for pain management, this type of receptor regulation can be challenging. One of the therapeutic strategies to potentiate MOP agonist activity has been the addition of a CCK2
receptor antagonist, although the molecular and cellular basis for this is not yet clear. In a recent report, we developed a series of bivalent ligands that incorporate a spacer to link a MOP agonist with a CCK2
Those ligands included spacers of adequate length to be able to simultaneously bind to both receptors, inducing association of these two receptors. Although these bivalent ligands were not any more effective in producing antinocioception than the combination of the two monovalent ligands, the impact of such agents on receptor internalization has not previously been studied.
Receptor antagonists, unlike agonist ligands, often do not elicit any regulatory response to their binding to their receptors. There are some examples of antagonists stimulating receptor internalization,10,11
but these tend to represent relatively rare exceptions. Similarly, some ligands previously believed to represent antagonists have actually turned out to be biased agonists, eliciting non-G protein-mediated signaling events, often mediated by arrestin-like protein interactions with their receptors.12,13
In the current report, it is well demonstrated that the CCK2
receptor antagonist component of the bivalent agents studied does not stimulate receptor internalization, and it was previously shown to not elicit any signaling response in receptor-bearing cells.9
As is often the case, the MOP agonist pharmacophore incorporated into the bivalent ligand stimulated the internalization of its receptor in a time- and temperature-dependent manner. The pattern of internalization was quite typical of receptor-mediated endocytosis that is so common after agonist stimulation of G-protein-coupled receptors.14
Perhaps the most interesting feature of this work is what the effect might be on the internalization of both the MOP receptor and the CCK2
receptor after their occupation with the bivalent ligands. The bivalent ligand with the short nine-atom spacer that does not allow simultaneous binding to both receptors stimulated internalization of the MOP receptor but not internalization of the CCK2
receptor. The bivalent peptide ligand that was previously reported (7
had a similar effect on these receptors. Without existing precedent, it was equally plausible that simultaneous occupation of both the MOP and the CCK2
receptors with a tethering bivalent ligand could interfere with the internalization of the MOP receptor by being retained on the cell surface or could stimulate internalization of the CCK2
receptor. Indeed, the latter turned out to represent the dominant action. All three bivalent ligands that were capable of bridging the MOP and CCK2
receptors had the effect of stimulating the internalization of both MOP and CCK2
receptors in a time- and temperature-dependent manner.
Since receptor internalization induced by the bivalent ligands prevailed over the tendency for the second receptor to remain on the cell surface, this effect might have the expected impact of down-regulating the CCK2 receptor and the biological effects of subsequent stimulation with CCK2 receptor agonists. This could also theoretically result in making such bivalent ligands less effective than two monovalent ligands. The other interesting extension of these observations is the possibility of targeted “dragging” of a receptor from the cell surface without the presence of an agonist for that receptor in order to induce its down-regulation. This might represent a novel strategy to regulate a receptor-mediated process.