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Functional characterization of putative cholesterol binding sequence (CRAC) in human type‐1 cannabinoid receptor
Author(s) -
Oddi Sergio,
Dainese Enrico,
Fezza Filomena,
Lanuti Mirko,
Barcaroli Daniela,
De Laurenzi Vincenzo,
Centonze Diego,
Maccarrone Mauro
Publication year - 2011
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2010.07041.x
Subject(s) - biology , receptor , peptide sequence , consensus sequence , amino acid , biochemistry , cannabinoid , cannabinoid receptor , transmembrane domain , serine , microbiology and biotechnology , gene , agonist , enzyme
J. Neurochem. (2011) 116 , 858–865. 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.