Modulation of Cell Surface Expression of Nonactivated Cholecystokinin Receptors Using Bivalent Ligand-Induced Internalization

作者：Harikumar Kaleeckal G; Akgun Eyup; Portoghese Philip S; Miller Laurence J^*

来源：Journal of Medicinal Chemistry, 2010, 53(7): 2836-2842.

DOI：10.1021/jm100135g

CCK(2) receptor antagonists potentiate pain relief by MOP receptor agonists. In an attempt to enhance this effect, we prepared bivalent ligands incorporating CCK, 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 nonfluoreseent 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.

• 出版日期2010-4-8