Pharmacological comparison of a recombinant CB1 cannabinoid receptor with its G(alpha 16) fusion product.
The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB(1) agonist-mediated signal transduction via an engineered G(alpha 16) system is different than that of the G(i/o) coupling normally preferred by the CB(1) receptor, we transfected the human recombinant CB(1) receptor (hCB(1)) or a fusion protein comprising the hCB(1) receptor and G(alpha 16) (hCB(1)-G(alpha 16)) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB(1)-G(alpha 16) fusion protein was found to be similar to that for hCB(1): HU 210 > CP 55,940 > or = SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [(35)S]GTP gamma S binding or inhibited forskolin-stimulated cAMP, presumably by coupling to G(i/o), in cells expressing hCB(1) but not hCB(1)-G(alpha 16). However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCB(1)-G(alpha 16) but not hCB(1). These data demonstrate that ligand affinities for the hCB(1) receptor are not affected by fusion to the G(alpha 16) subunit. Furthermore, there is essentially no difference in the function of the hCB(1) receptor when coupled to G(i/o) or G (alpha 16).
Martin
Renée S
RS
CNS Therapy Area, Neurobiology Unit, Roche Bioscience, Palo Alto, CA 94304, USA. renee.martin@roche.com
Reynen
Paul H
PH
Calixto
Joyce J
JJ
Reyes
Christian L
CL
Chang
Thomas K
TK
Dietrich
Paul S
PS
Bonhaus
Douglas W
DW
MacLennan
Stephen J
SJ
D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000032:analysis; D003511:Cyclohexanols / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000494:pharmacology*; D006801:Humans; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D013462:Sulfur Radioisotopes / Q000378:metabolism; D014316:Tritium / Q000378:metabolism
D003511:Cyclohexanols; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D000242:Cyclic AMP; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium