Induced association of mu opioid (MOP) and type 2 cholecystokinin (CCK2) receptors by novel bivalent ligands

Abstract

Both mu-opioid (MOP) and type 2 cholecystokinin (CCK2) receptors are present in areas of the central nervous system that are involved in modulation of pain processing. We conducted bioluminescence resonance energy transfer (BRET) studies on COS cells coexpressing MOP and CCK2 receptors to determine whether receptor heterodimerization is involved in such modulation. These studies revealed the absence of constitutive or monovalent ligand-induced heterodimerization. Heterodimerization of MOP and CCK2 receptors therefore is unlikely to be responsible for the opposing effects between morphine and CCK in the CNS. However, association was induced, as indicated by a positive BRET signal, on exposure of the cells to bivalent ligands containing mu-opioid agonist and CCK2 receptor antagonist pharmacophores linked through spacers containing 16-22 atoms but not with a shorter (9-atom) spacer. These studies demonstrate for the first time that an appropriately designed bivalent ligand is capable of inducing association of G-protein-coupled receptors. The finding that opioid tolerance studies with these ligands in mice showed no correlation with the BRET data is consistent with the absence of association of MOP and CCK2 receptors in vivo.

Figure 1

Opioid agonist 1 and CCK₂ receptor antagonist 2 pharmacophores and related bivalent 3a-c, 4 and monovalent 5, 6 ligands.

Scheme 1

a) i. (1S)-(+)-10-camphorsulfonic acid, CH₃CN/diethylether; ii. 10% NaOH, CH₂Cl₂. b) Mother liquor from first step taken. i. (1R)-(−)-10-camphorsulfonic acid, CH₃CN/diethyl ether; ii. 10% NaOH, CH₂Cl₂. c) Triphosgen, aquas sat. Na₂CO₃/CH₂Cl₂, 0 °C.

Scheme 2

a) HOBt/DCC, DMF, Bochexadiamine1,6, r.t.; b) TFA/CH₂Cl₂, r.t.; c) only 12 used, HOBt/DCC, DMF, r.t.: d) Pd/C (10 %), methanol,50 psi.; e) 8, DMF, 5 days.

Scheme 3

a) Diglycolic anhydride, CH₂Cl₂, r.t.; b) HOBt/DCC, mono-Boc diamines (n=2; n=6), DMF; c) TAF/CH₂Cl₂; d) 12, HOBt/DCC; e) Pd/C (10%), methanol, 50 psi; f) 8, DMF, ∼24h, r.t.

Scheme 4

a) HOBt/DCC, DMF; b) Pd/C (10%), methanol, 50 psi; c) 8, DMSO, 24h, r.t.

Figure 2. BRET controls

Shown are the BRET ratios for COS cells expressing various constructs in the combinations noted. The shaded area represents the intensity of BRET signals felt to be non-specific, reflecting a signal that can be generated between Rlu-tagged receptor and soluble YFP protein or between YFP-tagged receptor and soluble Rlu protein. BRET signals above this are considered to be significant. Both CCK₂ receptors and MOP receptors exist as constitutive homodimers with significant homologous receptor BRET signals when expressed in these cells. Values presented represent means±S.E.M. of data from five independent experiments. Values marked with ** represent BRET signals significantly above non-specific values at the p<0.001 level.

Figure 3. Effects of bivalent ligands on heterodimerization of CCK₂ and MOP receptors

Shown are BRET ratios for COS cells expressing both tagged MOP and CCK₂ receptor constructs with or without incubation of various bivalent ligands (10⁻⁶ M, 90 min at room temperature). The panels on the left reflect Rlu-tagged mu-opioid receptor expressed with YFP-tagged CCK₂ receptor, while those on the right represent the opposite. The panels in the top row illustrate BRET ratios for these cells in the absence of ligand and in the presence of bivalent ligands of various spacer lengths. The panels in the second row reflect the saturability of the BRET ratios in the presence of the bivalent ligands that normally give a significant signal, that are reduced in the presence of competing monovalent CCK antagonist ligand 6. The panels in the third row demonstrate that monovalent ligands that recognize only the CCK₂ receptor or the opioid receptor, even when mixed together, failed to produce the BRET signal observed with the bivalent ligand. The shaded area represents the non-specific BRET signal as described in Figure 2. Values represent means±S.E.M. of data from five independent experiments. * p<0.05, ** p<0.001.

Figure 3. Effects of bivalent ligands on heterodimerization of CCK₂ and MOP receptors

Shown are BRET ratios for COS cells expressing both tagged MOP and CCK₂ receptor constructs with or without incubation of various bivalent ligands (10⁻⁶ M, 90 min at room temperature). The panels on the left reflect Rlu-tagged mu-opioid receptor expressed with YFP-tagged CCK₂ receptor, while those on the right represent the opposite. The panels in the top row illustrate BRET ratios for these cells in the absence of ligand and in the presence of bivalent ligands of various spacer lengths. The panels in the second row reflect the saturability of the BRET ratios in the presence of the bivalent ligands that normally give a significant signal, that are reduced in the presence of competing monovalent CCK antagonist ligand 6. The panels in the third row demonstrate that monovalent ligands that recognize only the CCK₂ receptor or the opioid receptor, even when mixed together, failed to produce the BRET signal observed with the bivalent ligand. The shaded area represents the non-specific BRET signal as described in Figure 2. Values represent means±S.E.M. of data from five independent experiments. * p<0.05, ** p<0.001.

Figure 4. Effects of ligands on receptor homodimerzation

Shown are the BRET signals stimulated by bivalent or monovalent ligands in COS cells expressing Rlu- and YFP-tagged CCK₂ receptor constructs or MOP receptor constructs or structurally-unrelated tagged receptor constructs. None of the ligands studied had any effect on the constitutive homodimers of the CCK₂ or MOP receptors, and did not lead to heterodimerization of the unrelated receptors. Values represent means±S.E.M of data from six independent experiments. * p<0.05

Figure 5. Effects of bivalent ligands on bystander BRET

Shown are the saturation curves obtained by co-expressing pairs of Rlu-tagged MOP receptor and YFP-tagged CCK₂ receptor constructs after incubation with bivalent ligands (10⁻⁶M), as indicated. Bivalent ligands 3a-c produced exponential curves that reached a plateau, supporting specific molecular interactions. Cells treated with either no ligand or with a combination of monovalent ligands for the CCK and mu-opioid receptors showed curves not significantly different from linear fits. Values represent means±S.E.M of data from five independent experiments.

Figure 6

Schematic illustration of the effect of a MOP agonist/CCK₂ antagonist bivalent ligand on the equilibrium between a mixture of MOP receptor (MOPR) and cholecystokinin receptors (CCK₂R). Note that the bivalent ligand induces heterodimerization through constraint imposed by its spacer.