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 five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define 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 wild-type 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. 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 five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define 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 wild-type 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 fluidity, as well as its dimensions (1.Brown D.A. London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts.J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2056) Google Scholar, 2.Edidin M. Shrinking patches and slippery rafts: scales of domains in the plasma membrane.Trends Cell Biol. 2001; 11: 492-496Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 3.Pike L.J. Lipid rafts: bringing order to chaos.J. Lipid Res. 2003; 44: 655-667Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar). It has recently become clear that cholesterol can be tightly associated with membrane proteins, including G protein-coupled receptors (GPCRs) (4.Barrantes F.J. Cholesterol effects on nicotinic acetylcholine receptor: cellular aspects.Subcell. Biochem. 2010; 51: 467-487Crossref PubMed Scopus (40) Google Scholar, 5.Gimpl G. Fahrenholz F. Human oxytocin receptors in chol