Cholesterol residence time drives regulation of the G protein-coupled cholecystokinin receptor

Membrane cholesterol modulates the ability of cholecystokinin to act at the type 1 cholecystokinin receptor (CCK1R), with elevated levels of this lipid that can be present in obesity, disrupting stimulus-activity coupling. This occurs via direct effect on a cholesterol association (cholesterol recognition/interaction amino acid consensus) motif within CCK1R, whereas an analogous motif is present in the type 2 cholecystokinin receptor (CCK2R) with no functional impact of cholesterol on that receptor. We recently demonstrated that surface residues surrounding this motif in these 2 G protein-coupled receptors could be exchanged as a group to convert their cholesterol sensitivity. We now evaluate each of these residues individually and use molecular dynamics to study cholesterol interactions with this site. These studies show that cholesterol occupies this motif in both CCK1R and CCK2R with similar dominant poses in both receptors in normal, as well as in elevated cholesterol environments. The dominant pose of cholesterol changes in both receptors with elevated cholesterol, shifting proximity from transmembrane segment (TM) 3 toward transmembrane segment 5. Of note, cholesterol residence time at this motif is longer for CCK2R than CCK1R and longer in high cholesterol conditions at both receptors. This suggests that the cholesterol sensitivity of CCK1R is likely a dynamic event mediated by short, transient cholesterol interaction with CCK1R under normal conditions. One residue in position 5.52 was critical for this cholesterol effect on CCK1R, despite the prediction that this is not due to direct interaction with cholesterol. Instead, the packing of hydrophobic residues around 5.52 is tighter in CCK1R than in CCK2R, likely affecting the position and movement of transmembrane segment 5 that are important for stimulus-activity coupling. SIGNIFICANCE STATEMENT: Membrane cholesterol can affect the function of select G protein-coupled receptors, such as the type 1 cholecystokinin receptor, that are targets for drugs in disease states in which membrane cholesterol can be elevated. This article studied the molecular basis for high cholesterol disruption of stimulus-activity coupling at this receptor using site-directed mutagenesis and molecular dynamics. The findings demonstrate that cholesterol residence time at a key cholesterol association motif within CCK1R is responsible for this effect.