TY - JOUR
T1 - Functional characterization of putative cholesterol binding sequence (CRAC) in human type-1 cannabinoid receptor
AU - Oddi, Sergio
AU - Dainese, Enrico
AU - Fezza, Filomena
AU - Lanuti, Mirko
AU - Barcaroli, Daniela
AU - De Laurenzi, Vincenzo
AU - Centonze, Diego
AU - MacCarrone, Mauro
PY - 2011/3
Y1 - 2011/3
N2 - Endocannabinoid signaling modulates a variety of neuroinflammatory and neurodegenerative diseases, mainly through the activation of type-1 and type-2 (CB1R and CB2R) cannabinoid receptors. CB1R is negatively regulated by membrane cholesterol, while CB2R 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 CB1R into glycine, to obtain a cholesterol recognition/interaction amino acid sequence and consensus similar to that of CB2R. 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 CB1R 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.
AB - Endocannabinoid signaling modulates a variety of neuroinflammatory and neurodegenerative diseases, mainly through the activation of type-1 and type-2 (CB1R and CB2R) cannabinoid receptors. CB1R is negatively regulated by membrane cholesterol, while CB2R 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 CB1R into glycine, to obtain a cholesterol recognition/interaction amino acid sequence and consensus similar to that of CB2R. 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 CB1R 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.
KW - cannabinoid receptor
KW - cholesterol
KW - CRAC
KW - FRAP
KW - mutagenesis
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UR - http://www.scopus.com/inward/citedby.url?scp=79851494039&partnerID=8YFLogxK
U2 - 10.1111/j.1471-4159.2010.07041.x
DO - 10.1111/j.1471-4159.2010.07041.x
M3 - Article
C2 - 21214565
AN - SCOPUS:79851494039
VL - 116
SP - 858
EP - 865
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
SN - 0022-3042
IS - 5
ER -