Functional consequences of nonsynonymous single nucleotide polymorphisms in the CB2 cannabinoid receptor.
OBJECTIVE
To test the hypothesis that the two nonsynonymous single nucleotide polymorphisms at the CB2 cannabinoid receptor gene may have functional consequences on human CB2.


METHODS
Q63R, H316Y, and Q63R/H316 mutations were made in recombinant human CB2 by the method of site-directed mutagenesis. After these mutant CB2 receptors were stably transfected into HEK293 cells, ligand binding, ligand-induced activity, and constitutive activity assays were performed to test the functional significance of these mutations.


RESULTS
In general, our results showed that the CB2 polymorphic receptors are able to bind cannabinoid ligands and mediate signal transduction. However, in ligand-induced cyclic AMP accumulation assays, the cannabinoid agonists WIN55212-2 and 2-arachidonoylglycerol had reduced efficacy in cells expressing the polymorphic receptors as compared with the CB2 wild-type receptor. Furthermore, in constitutive activity assays, the H316Y and Q63R/H316Y polymorphic receptors exhibited higher constitutive activity than the CB2 wild-type receptor.


CONCLUSIONS
Our data shows that the presence of the polymorphisms at both positions 63 and 316 produce alterations in the CB2 receptor functions. Moreover, these findings strengthen the idea that the CB2 polymorphic receptors may contribute to the etiology of certain diseases.
Carrasquer
Alex
A
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA.
Nebane
Nstang M
NM
Williams
Walter M
WM
Song
Zhao-Hui
ZH
D000483:Alleles; D019943:Amino Acid Substitution; D001095:Arachidonic Acids / Q000819:agonists; D001120:Arginine / Q000378:metabolism; D048588:Benzoxazines / Q000819:agonists; D002186:Cannabinoids / Q000819:agonists; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D063388:Endocannabinoids; D005787:Gene Frequency; D005989:Glycerides / Q000819:agonists; D006801:Humans; D007668:Kidney / Q000166:cytology; D008024:Ligands; D009025:Morpholines / Q000819:agonists; D009154:Mutation; D009281:Naphthalenes / Q000819:agonists; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding / Q000235:genetics; D043885:Receptor, Cannabinoid, CB2 / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D001095:Arachidonic Acids; D048588:Benzoxazines; D002186:Cannabinoids; D063388:Endocannabinoids; D005989:Glycerides; D008024:Ligands; D009025:Morpholines; D009281:Naphthalenes; D043885:Receptor, Cannabinoid, CB2; D014443:Tyrosine; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; C094503:glyceryl 2-arachidonate; D001120:Arginine; D000242:Cyclic AMP