Functional characterization of putative cholesterol binding sequence (CRAC) in human type-1 cannabinoid receptor

Endocannabinoid signaling modulates a variety of neuroinflammatory andneurodegenerative 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 bymembrane cholesterol, while CB(2)R is unaffected. Here, we identified in thetransmembrane helix 7 of human CBRs a consensus sequence already known in otherproteins as cholesterol recognition/interaction amino acid sequence and consensuspattern. As this motif is different in the two CBR subtypes, we mutated lysine402 of CB(1)R into glycine, to obtain a cholesterol recognition/interaction aminoacid sequence and consensus similar to that of CB(2)R. Both mutated and wild-typereceptors were transiently expressed in human neuronal SH-SY5Y cells, and theirlocalization and functioning were investigated using biochemical assays andimmunofluorescence labelling. We found a reduced propensity of the mutant CB(1)R to reside in cholesterol-rich microdomains and, by means of fluorescence recoveryafter photobleaching analysis, we documented its loss of sensitivity to increasedmembrane cholesterol content. These results seem to uncover the existence of anew structural determinant in cannabinoid receptors, that is likely implicated indirecting their interaction with cholesterol-rich microdomains of cell membranes.[...]