Modulation of cell surface expression of nonactivated cholecystokinin receptors using bivalent ligand-induced internalization

Abstract

CCK(2) receptor antagonists potentiate pain relief by MOP receptor agonists. In an attempt to enhance this effect, we prepared bivalent ligands incorporating CCK(2) receptor antagonist and MOP receptor agonist pharmacophores. (9) Ligands with 16- to 22-atom spacers could simultaneously bind both receptors but provided no advantage in activity over individual ligands. We now examine the effect of these ligands on receptor internalization as a mechanism of receptor regulation. We prepared CHO cell lines expressing nonfluorescent halves (YN and YC) of yellow fluorescent protein attached to each receptor. Spatial approximation of constructs was needed to yield fluorescence. Monovalent MOP agonist 1 signaled normally and internalized the MOP receptor. Monovalent CCK(2) antagonist 2 did not stimulate receptor internalization. In the dual receptor-bearing cells, bivalent ligands 3a-c capable of simultaneously binding both receptors resulted in cell surface fluorescence and internalization of the fluorescent complex in a time- and temperature-dependent manner. Bivalent ligand 4 with spacer too short to occupy both receptors simultaneously yielded no signal. Receptor tethering with appropriate bivalent ligands can down-regulate signaling by moving a nonactivated receptor into the endocytic pathway.

Figure 1

Structures of monovalent and bivalent ligands. Shown are the chemical structures of the monovalent ligands with MOP (1) or CCK₂ (2) receptor pharmacophores and bivalent ligands (3a–c, 4) containing both pharmacophores.

Figure 2

Bimolecular fluorescence complementation. Shown are representative epifluorescence images of COS cells expressing the indicated receptor constructs. Microscopic images show the effective physical complementation of YN-tagged receptor and YC-tagged receptor to yield fluorescent YFP. These observations support the previous reports of constitutive homo-oligomerization of the CCK₂ and MOP receptors. No YFP fluorescence complementation was observed when the complementarily tagged CCK₂ and MOP receptors were coexpressed, again supporting the previous BRET study showing absence of hetero-oligomerization. Scale bar: 25 μm.

Figure 3

Receptor expression on CHO-CCK₂-YN/MOP-YC cells. Shown are concentration–response curves in intact cells, which represent compound 5 competing for CCK radioligand binding (A) and compound 6 inhibiting forskolin-stimulated cAMP responses (B). Data are expressed as the mean ± SEM of values from three to four independent experiments performed in duplicate.

Figure 4

Ligand-dependent internalization of MOP receptors and CCK₂ receptors on CHO cell lines. Shown are representative epifluorescence microscopic images illustrating time-dependent internalization of YFP-tagged MOP receptor exposed to compound 6 (A) and absence of internalization of the YFP-tagged CCK₂ receptor exposed to compound 5 (B). In contrast, CCK stimulation of the CCK₂ receptor is known to stimulate receptor internalization. Also shown are similar images from these cells stimulated with the monovalent MOP receptor agonist (compound 1) (C) and monovalent CCK₂ receptor antagonist (compound 2) (D). Scale bar: 25 μm.

Figure 5

Bivalent ligand-induced fluorescence complementation in CHO-CCK₂-YN/MOP-YC cells. Shown are representative phase contrast and epifluorescence microscopic images of CHO-CCK₂-YN/MOP-YC cells exposed to various ligands for 90 min at 4 °C. Compounds 1 and 2, representing monovalent ligands, were unable to form intact fluorescent YFP. Compounds 3a, 3b, and 3c, representing bivalent ligands with adequate length spacers to bind to both receptors simultaneously, were able to form intact fluorescent YFP on the cell surface under these conditions. Compound 4, with the short nine-atom spacer, and the bivalent peptidyl ligand (Tyr-D-Ala-Gly-D-Trp-NMe-Nle-Asp-Phe-NH₂, compound 7) were not able to form fluorescent YFP. Scale bar: 25 μm.

Figure 6

Time-dependent internalization of CCK₂ and MOP receptors in CHO-CCK₂-YN/MOP-YC cells. Bivalent ligands, compounds 3a, 3b, and 3c, that were able to elicit fluorescent YFP by complementation of the tags on the CHO-CCK₂-YN/MOP-YC cells also stimulated the time-dependent internalization of these complexes. Scale bar: 25 μm.