443 0026-895X/95/030443-08$3.O0/0 Copyright © by The American 5ociety for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 48:443-450 (1995). Comparison of the Pharmacology and Signal Transduction of the Human Cannabinoid CB1 and CB2 Receptors CHRISTIAN C. FELDER, KELLY E. JOYCE, EILEEN M. BRILEY, JALEH MANSOURI, KEN MACKIE, OLIVIER BLOND, YVONNE LAI, ALICE L. MA, and RICHARD L. MITCHELL Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892 (C.C.F., K.E.J., E.M.B.), Department of Physiology and Anesthesiology, University of Washington, Seattle, Washington 98195 (K.M., O.B.), Panlabs Inc., Seattle, Washington 98195 (Y.L., R.L.M.), and Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, Ma,yland 20892 (J.M.) Received April 1, 1995; Accepted June 20, 1995 SUMMARY The recently cloned GB2 cannabinoid receptor subtype was stably transfected into AtT-20 and Chinese hamster ovary cells to compare the binding and signal transduction properties of this receptor with those of the CB, receptor subtype. The binding of rH]CP 55,940 to both CB1 and CB2 was of similar high affinity (2.6 and 3.7 rlM, respectively) and saturable. In competitive binding experiments, (-)-9-tetrahydrocannabinol and CP 55,940 were equipotent at the CB1 and CB2 receptors, but WIN 55212-2 and cannabinol bound with higher affinity to the CB2 than the CB1 receptor. HU 21 0 had a higher affinity for the CB1 receptor. Anandamide, a recently identified endoge- nous cannabinoid agonist, was essentially equipotent at both receptor subtypes. The structurally related fatty acid ethanol- amides dihomo-y-Iinolenylethanolamide and mead ethanol- amide also bound with relatively equal affinity to both recep- tors, but adrenylethanolamide had a higher affinity for the CB1 receptor. The rank order of potency and efficacy for binding of the selected agonists to the GB1 and GB2 receptors was mim- icked in functional inhibition of cAMP accumulation experi- ments for all compounds tested. Both CB1 and CB2 receptors couple to the inhibition of cAMP accumlation that was pertussis toxin sensitive. SRi 41 71 6A, a CB1 receptor antagonist, was a poor antagonist at the CB2 receptor in both binding and func- tional inhibition of cAMP accumlation experiments. When ex- pressed in AtT-20 cells, the CB1 receptor mediated an inhibition of Q-type calcium channels and an activation of inward recti- fying potassium channels. In contrast, the GB2 receptor did not modulate the activity of either channel under identical assay conditions. Similar to results obtained for CB1 receptor, the CB2 receptor did not couple to the activation of phospholipases A2, C, or D or to the mobilization of intracellular Ca2 . Except for its inability to couple to the modulation of Q-type calcium channels or inwardly rectifying potassium channels, the CB1 and GB2 receptors display similar pharmacological and bio- chemical properties. The cannabinoids, which include (-)-z9-THC, the active principle in marijuana, are a large and diverse group of lipophilic compounds that mediate both psychoactive and systemic physiological effects such as analgesia, anticonvul- sion, immunosuppression, and the alleviation ofboth intraoc- ular pressure and emesis (1). The cannabinoids exert their effects by binding selectively to G protein-coupled cannabi- noid receptors located in the plasma membrane (2). Two subtypes of cannabinoid receptor have been cloned, CB1 (3) This project was funded in part by SBIR Phase I Grant No. 1 R43DA09203-0l to Panlabs, Inc., by The Keck Foundation, by a McKnight Research Award, and by National Institutes of Health Grants NS01588, DA08934 and NS08174. and CB2 (4). The CB1 receptor is mainly distributed in the central nervous system, with the highest levels found in the cerebellum, hippocampus, and striatum (5-7) and lesser amounts found in the testis (8). The CB2 receptor has only recently been cloned, and preliminary studies show its dis- tribution to be predominantly in peripheral tissues, including the marginal zone of the spleen, and in macrophages (4). Previous pharmacological studies established the presence of cannabinoid agonist binding sites in the central nervous system (9) and their functional coupling to the inhibition of both adenylate cyclase (10) and N-type voltage-dependent calcium channels (ii, 12). Cannabinoid agonists can also stimulate arachidonic acid release and inhibit its uptake (13, 14), although cannabinoid receptor involvement was not fully ABBREVIATIONS: (-)-9-THC, (-)-9-tetrahydrocannabinoI; arachidonic acid, eicosa-5Z,8Z,liZ,i4Z,-tetraenoic acid (20:4, n-6); mead acid, eicosa-5Z,8Z,i iZ-tnenoic acid (20:3, n-9); dihomo-y-linolenic acid, eicosa-8Z, 1 iZ, i4Z-trienoic acid (20:3, n-6); adrenic acid, eicosa-7Z, iOZ, i3Z, i6Z-tetraenoic acid (22:4, n-6); anandamide, arachidonylethanolamide; adrenylethanolamide, docosatetraenylethanolamide; mead ethano- lamide, eicosatnenylethanolamide; CHO, Chinese hamster ovary; L, Ltk; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; TLC, thin layer chromatography; HEPES, 4-(2-hydroxyethyl)-i -piperazineethanesulfonic acid; EGTA, ethylene glycol bis(13-aminoethyl ether)- N,N,N’,N’-tetraacetic acid. at Indiana Univ Lib/Serials Dept/Attn: M Grubaugh on August 14, 2008 molpharm.aspetjournals.org Downloaded from