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

Sergio Oddi, Enrico Dainese, Filomena Fezza, Mirko Lanuti, Daniela Barcaroli, Vincenzo De Laurenzi, Diego Centonze, Mauro MacCarrone

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)858-865
Number of pages8
JournalJournal of Neurochemistry
Volume116
Issue number5
DOIs
Publication statusPublished - Mar 2011

Keywords

  • cannabinoid receptor
  • cholesterol
  • CRAC
  • FRAP
  • mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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