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

  1. Sergio Oddi1,2,†,
  2. Enrico Dainese1,†,
  3. Filomena Fezza2,3,
  4. Mirko Lanuti1,2,
  5. Daniela Barcaroli4,
  6. Vincenzo De Laurenzi4,
  7. Diego Centonze2,5,
  8. Mauro Maccarrone1,2

Article first published online: 7 JAN 2011

DOI: 10.1111/j.1471-4159.2010.07041.x

Issue

Journal of Neurochemistry

Journal of Neurochemistry

Volume 116, Issue 5, pages 858–865, March 2011

Additional Information

How to Cite

Oddi, S., Dainese, E., Fezza, F., Lanuti, M., Barcaroli, D., De Laurenzi, V., Centonze, D. and Maccarrone, M. (2011), Functional characterization of putative cholesterol binding sequence (CRAC) in human type-1 cannabinoid receptor. Journal of Neurochemistry, 116: 858–865. doi: 10.1111/j.1471-4159.2010.07041.x

Author Information

  1. 1

    Department of Biomedical Sciences, University of Teramo, Teramo, Italy

  2. 2

    European Center for Brain Research (CERC)/Santa Lucia Foundation I.R.C.C.S., Rome, Italy

  3. 3

    Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Rome, Italy

  4. 4

    Department of Biomedical Sciences, University of Chieti-Pescara ‘G. d’Annunzio’, Chieti, Italy

  5. 5

    Department of Neurosciences, University of Rome ‘Tor Vergata’, Rome, Italy

  1. †

    These authors contributed equally to this study.

*Address correspondence and reprint requests to Prof. Mauro Maccarrone, Department of Biomedical Sciences, Piazza A. Moro 45, I-64100 Teramo, Italy. E-mail: mmaccarrone@unite.it

Publication History

  1. Issue published online: 9 FEB 2011
  2. Article first published online: 7 JAN 2011
  3. Received July 29, 2010; revised manuscript received September 24, 2010; accepted September 30, 2010.

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Keywords:

  • cannabinoid receptor;
  • cholesterol;
  • CRAC;
  • FRAP;
  • mutagenesis

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 (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.

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