Abstract

CB₁ is a G protein coupled receptor (GPCR) abundant in the central nervous system. It binds several compounds in its orthosteric site, including the endocannabinoids, arachidonoyl ethanolamide (anandamide; AEA) and 2-arachidonoyl glycerol (2-AG), and the plant-derived Δ^9-tetrahydrocannabinol (THC), one of the main psychoactive components of marijuana. It primarily couples to G_i/o proteins to inhibit adenylate cyclase activity and typically induces downstream signaling that is G_i-dependent. Since this receptor is implicated in several maladies, such as obesity, pain, and neurodegenerative disorders, there is interest in developing therapeutics that selectively target this receptor. Allosteric modulators of CB₁ offer one new approach that has tremendous therapeutic potential. Here, we reveal receptor- and cellular-level properties consistent with receptor activation by a series of pyrimidinyl biphenylureases (LDK1285, LDK1288, LDK1305, and PSNCBAM1). This includes promoting binding of the agonist CP55940 with positive cooperativity and inhibiting binding of the inverse agonist SR141716A with negative cooperativity. Consistent with these findings, the allosteric modulators induced cellular internalization of the receptor and recruitment of β-arrestin 2 in HEK293 cells monitored with confocal and TIRF microscopy, respectively. These allosteric modulators, however, cause G protein-independent but β-arrestin 1-dependent phosphorylation of the downstream kinases, ERK1/2, MEK, and Src using immunoblotting studies. These results are consistent with the involvement of β-arrestin and suggest these allosteric modulators induce biased signaling.