title: Functional characterization of putative cholesterol binding sequence (CRAC) in human type-1 cannabinoid receptor.
abstract: Endocannabinoid signaling modulates a variety of neuroinflammatory and neurodegenerative diseases, mainly through the activation of type-1 and type-2 (CB(1)R and CB(2)R) cannabinoid receptors. CB(1)R is negatively regulated by membrane cholesterol, while CB(2)R is unaffected. Here, we identified in the transmembrane helix 7 of human CBRs a consensus sequence already known in other proteins as cholesterol recognition/interaction amino acid sequence and consensus pattern. As this motif is different in the two CBR subtypes, we mutated lysine 402 of CB(1)R into glycine, to obtain a cholesterol recognition/interaction amino acid sequence and consensus similar to that of CB(2)R. Both mutated and wild-type receptors were transiently expressed in human neuronal SH-SY5Y cells, and their localization and functioning were investigated using biochemical assays and immunofluorescence labelling. We found a reduced propensity of the mutant CB(1)R to reside in cholesterol-rich microdomains and, by means of fluorescence recovery after photobleaching analysis, we documented its loss of sensitivity to increased membrane cholesterol content. These results seem to uncover the existence of a new structural determinant in cannabinoid receptors, that is likely implicated in directing their interaction with cholesterol-rich microdomains of cell membranes.
authors.affiliation: Department of Biomedical Sciences, University of Teramo, Teramo, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Oddi
authors.forename: Enrico
authors.initials: E
authors.lastname: Dainese
authors.forename: Filomena
authors.initials: F
authors.lastname: Fezza
authors.forename: Mirko
authors.initials: M
authors.lastname: Lanuti
authors.forename: Daniela
authors.initials: D
authors.lastname: Barcaroli
authors.forename: Vincenzo
authors.initials: V
authors.lastname: De Laurenzi
authors.forename: Diego
authors.initials: D
authors.lastname: Centonze
authors.forename: Mauro
authors.initials: M
authors.lastname: Maccarrone
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D016384:Consensus Sequence / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism; D045604:Extracellular Fluid / Q000187:drug effects / Q000378:metabolism; D005998:Glycine / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma / Q000473:pathology; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000379:methods
chemical_list: D043884:Receptor, Cannabinoid, CB1; D049452:Green Fluorescent Proteins; D002784:Cholesterol; D000242:Cyclic AMP; D008239:Lysine; D005998:Glycine