Functional mapping of cannabinoid receptor homologs in mammals, other vertebrates, and invertebrates

Over the past decade, several putative homologs of cannabinoid receptors (CBRs) have been identified by homology screening. Homology screening utilizes sequence alignment search engines to recognize homologs. We investigated these putative CBR homologs further by 'functional mapping' of their deduced amino acid sequences. The entire pharmacophore of a CBR has not yet been elucidated, but point-mutation studies have identified over 20 amino acid residues that impart CBR specificity for ligand recognition and/or signal transduction. Twenty point-mutation studies were used to construct a CBR functionality matrix. Sixteen putative CBR homologs were then mapped over the matrix. Several putative homologs did not hold up to this analysis: human GPR3, GPR6, GPR12, and Caenorhabditis elegans C02H7.2 expressed a series of crippling substitutions in the matrix, strongly suggesting they do not encode functional CBRs. Mapping the contested leech (Hirudo medicinalis) CBR sequence suggests that it encodes a functional CB1; it expresses fewer substitutions than the sea squirt (Ciona intestinalis) CB1 sequence. Mapping a putative CB2 ortholog in the puffer fish (Fugu rubripes T012234) suggests it may encode a CBR other than CB2. These findings are consistent with the lack of experimental data proving these putative CBRs have affinity for cannabinoid ligands. Matrix analysis also reveals that SR144528, a 'CB2-specific' synthetic antagonist, has affinity for non-mammalian CB1 receptors, and that L3.45 appears to be CB2-specific, its cognate in CB1 receptors is F3.45. In conclusion, functional mapping, utilizing point-mutation studies, may improve the specificity of homology screening performed by sequence alignment search engines.