Location of the cannabinoid CB1 receptor allosteric binding site

Gemma Baillie1, Teresa Barber3, Dow Hurst3, Mary Abood2, Patricia Reggio3. 1University of Aberdeen, Aberdeen, United Kingdom, 2California Pacific medical centre research group, San Francisco, United States, 3University of North Carolina, North Carolina, United States.

There is evidence for an allosteric binding site on the cannabinoid CB1 receptor (Price et al., 2005). The aim of this study was to identify the location of the allosteric site using single point amino acid mutations of the CB1 receptor.

The effect of the known allosteric inhibitor, ORG27569 (Price et al., 2005), on the efficacy of the CB1 receptor agonist CP55940 was investigated using the [35S]GTPγS binding assay (Thomas et al., 2005) in hCB1-HEK293 cells comparing wild type cells with cells mutated at W5.43A (Anavi-Goffer et al., 2007).

In cell lines expressing wild type CB1 receptors, CP55940 had an Emax of 21% (95% confidence limits, 14-28%) at a concentration of 10μM. In the presence of ORG27569 this stimulation was completely abolished. In comparison, using a W5.43A mutation, CP55940 had an Emax of 31% (95% confidence limits, 23-39%). In the presence of ORG27569 there was no significant change in the Emax which was 50% (95% confidence limits, 20-81%).

Thus, the mutation of the amino acid residue W5.43 resulted in a complete loss in the ability of ORG27569 to inhibit CP55940 allosterically. Previously this mutation was found to result in a loss of binding of the CB1 receptor inverse agonist, SR141716A (McAllister et al., 2003). These results indicate SR141716A and ORG27569 may bind to the cannabinoid CB1 receptor using a common amino acid residue.

Price, M.R et al. (2005) Mol Pharmacol 68, 1484-1495.
McAllister, S.D et al. (2003) J Biol Chem 279, 48024-48037.

Funded by NIH.