Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol

Abstract

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and fi ve. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to defi ne the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wildtype and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs. Cholesterol is an important lipid component of the eukaryotic plasma membrane that has substantial effects on the physicochemical characteristics of the membrane. These include effects on the membrane rigidity and fl uidity, as well as its dimensions ( 1-3 ).

Endogenous Allosteric Modulators of G Protein–Coupled Receptors

“…The affinities of ligands binding to the oxytocin receptor, cholecystokinin receptor 1 (CCK1), mGluR1a, and the 5-hydroxytryptamine 1A (5-HT 1A ) receptor are increased when these receptors are present in cholesterol-rich rafts compared with when they are expressed in cholesterol-depleted rafts (Gimpl et al, 1997;Eroglu et al, 2003;Prasad et al, 2009;Potter et al, 2012). Restoring the cholesterol content of cholesterol-depleted membranes increased the number of receptors in the lipid rafts and restored the binding affinity of the ligands to the receptors, in the case of oxytocin and mGluR1a receptors (Gimpl et al, 1997;Eroglu et al, 2003).…”

Cholecystokinin-induced satiety, a key gut servomechanism that is affected by the membrane microenvironment of this receptor

“…55,56 In these studies, the impact of the cholesterol was reproduced, with elevated cholesterol resulting in higher-affinity CCK binding and lower signaling responsiveness to CCK (Figure 1). 56 This was shown to be specific to the CCK1R, with the CCK2R being resistant to the deleterious effects of cholesterol.…”

“…55,56 In these studies, the impact of the cholesterol was reproduced, with elevated cholesterol resulting in higher-affinity CCK binding and lower signaling responsiveness to CCK (Figure 1). 56 This was shown to be specific to the CCK1R, with the CCK2R being resistant to the deleterious effects of cholesterol. 56 It is helpful to think about the CCK1R as having three dominant domains, the extracellular domain where the natural peptide ligand binds, the intracellular domain where the heterotrimeric G protein interacts with the receptor, and the intramembranous core helical bundle domain in which a network of hydrogen bonds helps to stabilize conformations and to mediate conformational changes associated with agonist binding and activation (Figure 2).…”

“…56 This was shown to be specific to the CCK1R, with the CCK2R being resistant to the deleterious effects of cholesterol. 56 It is helpful to think about the CCK1R as having three dominant domains, the extracellular domain where the natural peptide ligand binds, the intracellular domain where the heterotrimeric G protein interacts with the receptor, and the intramembranous core helical bundle domain in which a network of hydrogen bonds helps to stabilize conformations and to mediate conformational changes associated with agonist binding and activation (Figure 2). Clearly, the impact of the cholesterol is to cause a conformational change in this receptor that renders the latter domain dysfunctional.…”

“…Methods to increase the cholesterol in the cell membrane using methyl-β-cyclodextrin-cholesterol complex or LDL were established and applied to CCK1R-bearing cells. 56 Cells in which mutations in the lipid synthetic machinery were present and that resulted in elevated cholesterol were further engineered to express the CCK1R. 57 In addition, a CCK1R mutant affecting a residue at the bottom of TM3, near the DRY ionic lock mechanism and involving a cholesterol-binding motif, was shown to have all of the structural and functional effects typical of the wild-type CCK1R in a high-cholesterol environment.…”