CHARACTERIZATION OF 2 CLONED HUMAN CB1 CANNABINOID RECEPTOR ISOFORMS

We have investigated the pharmacology of two central human cannabinoid receptor isoforms, designated CB1 and CB1A, stably expressed in Chine se hamster ovary cell lines, designated as CHO-CB1 and CHO-CB1A, respe ctively. In direct binding assays on isolated membranes the agonist [H -3]CP 55,940 bound in a saturable and highly specific manner to both c annabinoid receptor isoforms. Competition binding experiments performe d with other commonly used receptor agonists showed the following rank order of potency: CP 55,940 > tetrahydrocannabinol > WIN 55212-2 > an andamide. Except for the endogenous ligand anandamide (CB1, K-i = 359. 6 nM vs. CB1A, K-i = 298 nM), these agonists bound to CB1A (CP 55,940, WIN 55212-2 and Delta(9)-THC, K-i = 7.24, 345 and 26.7 nM, respective ly) with about 3-fold less affinity than to CB1 (CP 55,940, WIN 55212- 2 and Delta(9)-THC, K-i = 2.26, 93 and 7.1 nM, respectively). The cann abinoid receptor antagonist SR 141716A also bound to CB1A (K-i = 43.3 nM) with slightly less affinity than to CB1 (K-i = 4.9 nM). Cannabinoi d receptor-linked second messenger system studies performed in the CHO -CB1 and CHO-CB1A cells showed that both receptors mediated their acti on through the agonist-induced inhibition of forskolin-stimulated cAMP accumulation. This activity was totally blocked by pretreatment with PTX. Additionally, both isoforms activated mitogen-activated protein k inase. The selective antagonist SR 141716A was able to selectively blo ck these responses in both cell lines, to an extent that reflected its binding characteristics. Our results show that the amino-truncated an d -modified CB1 isoform CB1A exhibits all the properties of CB1 to a s lightly attenuated extent.