Examination of the Effects of Cholesterol on Cholecystokinin Receptor Function using Model Cell Lines

Abstract

The Family A G protein-coupled type 1 cholecystokinin receptor (CCK1R) has been shown to be particularly sensitive to membrane cholesterol, with elevated levels induced by cholesterol-loaded methyl-β-cyclodextrin interfering with receptor-G protein coupling and hormone-induced signaling (JBC, 2005). This is important, because CCK is a physiologic satiety factor, and a defect in CCK-induced activation of this receptor can result in obesity with associated hyperlipidemia. These problems can then worsen the CCK signaling defect and contribute to a cycle of further clinical problems. Of note, the structurally closely-related CCK2R is not sensitive to membrane cholesterol when manipulated in the same manner (unpublished data). Here, we have attempted to study the function of these receptors in 25RA cells derived by T.Y. Chang from CHO cells (JBC 1980). These cells have elevated membrane cholesterol secondary to a gain-of-function defect in the SCAP gene (sterol regulatory element binding protein (SREBP) cleavage-activating protein). We established and characterized 25RA cell lines stably expressing CCK1R and CCK2R. Increased membrane cholesterol was visualized with a quantitative filipin-binding morphologic assay. In this cholesterol-rich environment, CCK radioligand binding and CCK-stimulated intracellular calcium assays were performed, with results compared to those in parental CHO cells. Like the CHO cells with acute physical enhancement of cholesterol, these genetically modified cells demonstrated defective CCK-induced signaling, despite normal CCK radioligand binding. We also analyzed CCK receptor trafficking in the environment of 25RA cells. We found that both CCK1R and CCK2R internalize in response to agonist occupation and to recycle to the plasma membrane normally despite the increase in membrane cholesterol.. (Supported by NIH grants DK32878 and DK78385, and a Kinney Career Development Award).