Abstract
Cannabinoid receptor 1 (CB1) is a G-protein–coupled receptor that is abundant in the central nervous system. It binds several compounds in its orthosteric site, including the endocannabinoids, arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol, and the plant-derived Δ9-tetrahydrocannabinol, one of the main psychoactive components of marijuana. It primarily couples to Gi/o proteins to inhibit adenylate cyclase activity and typically induces downstream signaling that is Gi-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 biphenylureas (LDK1285, LDK1288, LDK1305, and PSNCBAM1), including promoting binding of the agonist CP55940 with positive cooperativity and inhibiting binding of the inverse agonist SR141716A with negative cooperativity, demonstrated via radioligand binding studies. Consistent with these findings, the allosteric modulators induced cellular internalization of the receptor and recruitment of β-arrestin 2 in human embryonic kidney cell line 293 cells monitored with confocal and total internal reflective fluorescence microscopy, respectively. These allosteric modulators, however, caused G-protein–independent but β-arrestin 1–dependent phosphorylation of the downstream kinases extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Src, shown by immunoblotting studies. These results are consistent with the involvement of β-arrestin and suggest that these allosteric modulators induce biased signaling.
Footnotes
- Received April 27, 2018.
- Accepted October 12, 2018.
This work was supported in part by the National Institutes of Health, National Institute on Drug Abuse [Grant DA039942].
↵This article has supplemental material available at molpharm.aspetjournals.org.
- Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics