GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current

Abstract

The CB(1) cannabinoid receptor mediates many of the psychoactive effects of Delta(9)THC, the principal active component of cannabis. However, ample evidence suggests that additional non-CB(1)/CB(2) receptors may contribute to the behavioral, vascular, and immunological actions of Delta(9)THC and endogenous cannabinoids. Here, we provide further evidence that GPR55, a G protein-coupled receptor, is a cannabinoid receptor. GPR55 is highly expressed in large dorsal root ganglion neurons and, upon activation by various cannabinoids (Delta(9)THC, the anandamide analog methanandamide, and JWH015) increases intracellular calcium in these neurons. Examination of its signaling pathway in HEK293 cells transiently expressing GPR55 found the calcium increase to involve G(q), G(12), RhoA, actin, phospholipase C, and calcium release from IP(3)R-gated stores. GPR55 activation also inhibits M current. These results establish GPR55 as a cannabinoid receptor with signaling distinct from CB(1) and CB(2).

Fig. 1.

GPR55 activation by several cannabinoids increases intracellular calcium in HEK293 cells and DRG neurons. (A) The time course of changes in [Ca²⁺]_i in HEK293 cells loaded with fura-2 and perfused with THC. The lines above the figure indicate drug application. The solid line indicates a representative HEK293 cell transiently expressing the human GPR55 (hGPR55-HEK293) perfused with THC, whereas the black dashed line indicates a representative nontransfected HEK293 cell (HEK293 wt). (B) Summary of calcium responses upon perfusion of cannabinoid agonists. Responses in HEK293 wt cells (n = 6, black bars) and hGPR55-HEK293 cells (open bars) were perfused with THC, methanandamide (MEA), anandamide (AEA), or JWH015. (C) Immunostaining of mouse DRGs with a rabbit anti-GPR55 antibody (1:2,000, red) and a guinea pig anti-CB₁ antibody (1:2,000, green) show that GPR55 is preferentially expressed in large-diameter DRG neurons (arrowheads) whereas CB₁ is more widely expressed (arrows). See SI Text for full methods. (Scale bar, 50 μm.) (D) Representative JWH015-induced calcium rises in a large (solid line) and small (dashed line) DRG neuron. (E) Summary of calcium responses in small and large DRG neurons upon perfusion of THC, JWH015, and LPI.

Fig. 2.

The CB₁ antagonist SR141716A, but not the CB₂ antagonist SR144528, is a GPR55 antagonist. (A) Summary calcium responses in HEK293 wt cells or hGPR55-HEK293 cells treated with different combinations of agonist (THC, JWH015, or MEA) alone or coperfused with SR141716A (SR1). (B) Summary calcium responses in small or large DRG neurons treated with either THC or JWH015 alone or coperfused with SR1. (C) Summary calcium responses in HEK293 wt or hGPR55-HEK293 cells perfused with JWH015 or SR144528 (SR2) alone or together. (D) Summary calcium responses in small or large DRG neurons treated with JWH015 alone or coperfused with SR2.

Fig. 3.

GPR55 activation releases calcium from IP₃R-mediated calcium stores via phospholipase C activation. (A) A subset of hGPR55-HEK293 cells was perfused with either THC or JWH015 in 0 Ca²⁺ Ringer's solution, and the changes in [Ca²⁺]_i were compared with those in cells perfused with THC or JWH015 in control Ringer's solution (2 mM Ca²⁺). (B) Summary calcium responses in hGPR55-HEK293 cells pretreated with 1 μM thapsigargin (TG), or coperfused with either THC or JWH015, in combination with either the IP₃R inhibitor xestospongin D (XeD, 1 μM) or the RyR inhibitor dantrolene (Dan, 10 μM). These calcium rises were compared with those seen in control cells. (C) Summary calcium responses in large DRG neurons pretreated with 1 μM thapsigargin (TG) and perfused with THC. (D) Calcium changes in a subset of hGPR55-HEK293 cells treated with the PI-specific PLC inhibitor, ET-18-OCH₃ (edelfosine), relative to controls, upon perfusion of THC or JWH015.

Fig. 4.

G_q/11 is involved in the GPR55-induced increase in [Ca²⁺]_i. (A) The change in calcium upon THC application in a cell expressing hGPR55 alone or hGPR55 and dnG_αq. (B) Summary calcium changes in response to perfusion of either THC or JWH015 in HEK293 wt, hGPR55-HEK293, or hGPR55-HEK293 cells also expressing dnG_αq, dnG_α12, or dnRhoA or coperfused with latrunculin B.

Fig. 5.

GPR55 inhibits M-type potassium current. (A) Voltage activation of M current in a representative hGPR55-HEK293 cell expressing KCNQ2 and KCNQ3. Depolarization elicits noninactivating outward current with characteristic slow activation and deactivation. (B and C) Same cell as in A. (B) Deactivation protocol for M current before and during exposure to THC. (C) Time course of suppression of steady-state M current at −20 mV by repeated THC. (D) Summary of M current suppression by THC in cells expressing KCNQ2/KCNQ3 alone and in cells expressing hGPR55 and KCNQ2/KCNQ3.