The orphan receptor GPR55 is a novel cannabinoid receptor

Abstract

Background: The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes.

Experimental approach: Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways.

Key results: We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1.

Conclusions: These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Figure 1

Alignment between human (hGPR55), mouse (mGPR55) and rat (rGPR55) GPR55 protein sequences. The putative positions of the transmembrane regions (TM1-7), extracellular loops (EC1-3) and intracellular loops (EC1-3) are shown. The amino-acid differences in the previously published sequence (Sawzdargo et al., 1999) for human GPR55 at the IC2/TM4 boundary are shown above the sequences.

Figure 2

mRNA expression levels of GPR55 and CB₁ receptors in mouse tissues measured by quantitative PCR relative to m36B4. Tissues were dissected from C57BL/6 female mice. Samples from different mice were processed individually in all subsequent steps; RNA preparation, cDNA synthesis and quantitative PCR. Data are mean values±s.e.m. using tissues from eight (GPR55) or four mice (CB₁) and presented as per cent of the ubiquitously and homogenously expressed ribosomal protein 36B4.

Figure 3

Cell-surface expression of FLAG-tagged hGPR55. Immunofluorescence images of anti-FLAG-stained HEK293s cells transiently transfected with FLAG-hGPR55 (a) or empty vector (Vec.co; (c)). Corresponding phase-contrast images are shown in (b) and (d).

Figure 4

Radioligand binding to GPR55. Membranes prepared from cells transiently transfected with hGPR55 or vector control were incubated with 50 nM [³H]-CP55940, [³H]-SR141716 or [³H]-WIN55,212-2. Specific binding was determined by the addition of 10 μM unlabelled ligand as competitor. The bars show the specific binding (mean±s.e.m.; n=5) determined for each ligand.

Figure 5

(a) Concentration–response curves for various ligands at GPR55 determined using a GTPγS assay: (a) CP55940 and Δ⁹-THC; (b) cannabidiol antagonism of O1602 activation; (c) anandamide and WIN55,212-2; (d) O1602 and abnormal cannabidiol. Values shown are mean±s.e.m.; n=5.

Figure 6

Mapping G-protein coupling of GPR55. (a) Basal and 1 μM O1602 stimulated GTPγS binding (% activity, mean±s.e.m.) in human GPR55-expressing membranes in the absence and presence of various concentrations of peptides equivalent to the C termini of Gα₁₃, Gα_i1/2, Gα_i3 and Gα_s. (b) Basal and 1 μM stimulated GTPγS binding (% activity, mean±s.e.m.) in human GPR55-expressing membranes in the absence and presence of various dilutions of antibodies that bind to the C termini of Gα₁₃, Gα_i1/2, Gα_i3 and Gα_s. Data were analysed using paired t-test (^**P<0.05; ^***P<0.01; n=5).

Figure 7

Transfection of Gα₁₃ into GPR55-expressing HEK293 cells leads to an increased GTPγS signal via GPR55. Membranes prepared from HEK293s cells and HEK293s-GPR55-expressing cells were transfected with transfected with either control or Gα₁₃-containing plasmids and tested in a GTPγS with and without 1 μM O1602. Membranes containing GPR55 demonstrate a clear increase in GTPγS binding as a result of overexpression of Gα₁₃. Data (mean±s.e.m.) were analysed using paired t-test (^**P<0.05; n=5).

Figure 8

Activation of GPR55 leads to activation of rhoA, cdc42 and rac1. Cells transfected with GPR55 demonstrated O1602-(1 μM) and anandamide (1 μM)-mediated activation of the small G proteins rhoA, cdc42 and rac1 while the non-GPR55-activating ligand WIN55,212-2 had no effect. The activation was blocked by cannabidiol (10 μM) while the positive control GTPγS and negative controls (GDP and dimethyl sulphoxide ) generated the expected responses. The blots shown are representative of three independent experiments.