GPR55 as a New Cannabinoid Receptor: Still a Long Way to Prove It

A recent report indicates that GPR55 may be a new cannabinoid receptor sensitive to CP55940, a rather classical cannabinoid-like structure, and not to the alkylindole WIN55212-2 as previously thought (1). This analysis was made on the basis of sequence similarity between a number of GPR55 subdomains and the corresponding sequences of ‘classic‘ cannabinoid receptors, CB1 and CB2, and reinforced by the patent issued by AstraZeneca (2) showing that GPR55 is, indeed, pharmacologically a cannabinoid receptor. While the existence of additional cannabinoid receptors was suggested by several non-classical pharmacological responses (3–6), this was never clearly demonstrated. Mining our GPCR knowledge database (7), which includes biological activities related to mutagenesis experiments that have been collected and structured, we were able to determine an ensemble of amino acids residues, referred as ‘functional fingerprint’, involved in the binding and/or activity of a set of ligands potentially common to CB1, CB2 and GPR55. Ligands considered in this study were Δ9-tetrahydrocannabinol, CP55934, HU210, JWH051 and JWH057. Each of these compounds can be considered members of the classical cannabinoid family derived from natural terpenes. Rimonabant (SR141716), which is structurally quite dissimilar, was not considered albeit if the existence of hCB1-related mutagenesis experiments reported in the literature. Additional residues studied in molecular modelling with cannabinoid-like structures were considered. In the case of hCB2, and acknowledging the paucity of the literature on mutagenesis for this sequence, some residues modulating the binding of SR144528 or JWH015 were included. AurTAG is a semi-automatic annotation tool developed at Aureus (7), and it serves to retrieve all the amino acids for a given sequence involved in the regulation of binding or activity. By using AurTAG, we were able to define a functional fingerprint for both CB1 and hCB2 receptors (Table 1 and Figure 1). These annotations are based on the ratio between the values of key parameters of specific ligands. In the present study Ki, IC50 or EC50 from binding and second messenger-based activity tests were considered for a series of semi-synthetic cannabinoids. The hGPR55 sequence (Uniprot Q9Y2T6) was then aligned with hCB1 and hCB2 receptors (Uniprot P21554 and P34972, respectively) using ClustalW and the Blosum matrix in order to retrieve the hGPR55 amino acids aligned with those identified as key for the interaction of semi-synthetic cannabinoids with their ‘classical’ receptors. Schematic view of the strategy for defining cannabinoid receptor functional fingerprints from mutagenesis and molecular recognition literature data using AurTAG. Amino acid residues involved in binding or activity of “classical” cannabinoid ligands are retrieved from Aureus Pharma databases using AurTAG and define a functional fingerprint for hCB1 or hCB2 receptors. Amino acids constituting these fingerprints are poorly aligned in hGPR55 showing that this orphan receptor may not share the same binding pocket with already known cannabinoid receptor. The hCB2 receptor (Figure 1) only shares 44% homology with hCB1 receptor, however it exhibits a high homology to hCB1 functional fingerprint and hCB1 exhibits a high homology to hCB2 functional fingerprint. These relationships are in accordance with the fact that the two receptors probably share common features in their binding pocket for synthetic cannabinoids. In contrast, hGPR55 does not appear to share similar fingerprint with any of the classical CB receptors. Of course, this could not be considered as a proof for excluding a priori GPR55 from the CB family. But further work is needed to qualify and validate GPR55 as a cannabinoid receptor member. If it is confirmed that GPR55 is sensitive to some cannabinoid ligands and as GPR55 does not appear to have a classical ‘cannabinoid binding pocket’ it will be crucial to determine if these compounds could be allosteric ligands for GPR55.