Identification of G protein-coupled receptor 55 (GPR55) as a target of curcumin

Curcumin, a major bioactive compound in turmeric, exhibits various biological activities including anti-diabetic, anti-oxidative, antitumorigenic, anti-inflammatory, and neurotrophic properties. Its anti-diabetic effect is attributed to the induction of glucagon-like peptide-1 (GLP-1) secretion in intestinal L-cells. However, the underlying molecular mechanisms remain unclear. G protein-coupled receptors (GPCRs) are a large family of cell-surface receptors mediating responses to various stimuli and are important drug targets. While some food-derived small molecules, including polyphenols, are known to activate GPCRs, the specific ligand-receptor interactions are largely unknown. This study aimed to identify GPCRs as potential targets of curcumin and investigate the role of GPR55 in curcumin's physiological function.

Numerous studies have explored curcumin's diverse biological activities and its potential therapeutic applications. Its absorption, metabolism, and various biological activities are well-documented. The induction of GLP-1 secretion by curcumin and its implication in glucose homeostasis have been demonstrated in previous studies. However, the molecular mechanisms behind curcumin's effects, particularly its interaction with GPCRs, are not fully understood. The extensive family of GPCRs and their role in cellular signaling pathways have been comprehensively studied. Previous research has shown that some small molecules from food activate GPCRs, emphasizing the potential of GPCRs as targets for bioactive food compounds.

The researchers developed an expression screening system to identify GPCRs activated by curcumin. HEK293FT cells were transfected with GPCR expression vectors and a reporter vector. Curcumin's effect on reporter activity was measured after stimulating the cells. To determine the G protein involved, different reporter constructs mediating transcription via various G-protein pathways (G_s, G_i/o, G_q, and G_12/13) were used. The effects of curcumin-related compounds (demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin, curcumin B-D-glucuronide, and various metabolites and analogs) on GPR55 activation were also tested. The role of GPR55 in curcumin's activity was investigated using GPR55 antagonists (CID16020046). Structure-based docking simulations were performed using AutoDock Vina to identify key GPR55 residues involved in curcumin binding. Mutant GPR55 proteins were constructed to validate the docking results. The involvement of GPR55 in curcumin-induced GLP-1 secretion was studied using GLUTag enteroendocrine L-cells, measuring GLP-1 secretion and intracellular calcium levels in the presence and absence of a GPR55 antagonist. Western blotting was performed to analyze GPR55 protein levels after curcumin stimulation. Intracellular Ca²⁺ levels were measured using Fura-8 fluorescence. Statistical analysis included Student's t-test and one-way ANOVA.

The expression screening identified GPR55 as a potential target of curcumin. Curcumin activated GPR55 in a dose-dependent manner, inducing SRE- and SRF-RE-mediated transcription, indicative of G_12/13 signaling activation. The effects were comparable to those of LPI, an endogenous GPR55 ligand. Inhibitors of Rho kinase (Y-27632) but not MEK1/2 (U0126) suppressed curcumin-induced SRE-mediated transcription. Curcumin treatment decreased GPR55 protein levels, consistent with receptor internalization. Demethoxycurcumin also activated GPR55, while bisdemethoxycurcumin showed weaker activation. Tetrahydrocurcumin and curcumin B-D-glucuronide were inactive. Amongst the metabolites and analogs tested, only 2,6-dimethylcurcumin and 4',4''-dimethylcurcumin exhibited comparable GPR55 activation to curcumin. The GPR55 antagonist CID16020046 suppressed curcumin-induced SRE- and SRF-RE-mediated transcription, confirming curcumin's agonist role. Docking simulations predicted that F190^5.47, among other residues, is critical for curcumin binding to GPR55. Mutation of F190^5.47 to alanine significantly reduced curcumin's activation of GPR55, confirming its crucial role. Curcumin stimulated GLP-1 secretion in GLUTag cells, which was inhibited by CID16020046, further supporting the involvement of GPR55 in curcumin's anti-hyperglycemic effect. Curcumin also increased intracellular calcium levels in a GPR55-dependent manner.

The findings strongly suggest that curcumin acts as an agonist for GPR55, activating G_12/13 signaling pathways. This is consistent with previous research showing that GPR55 couples to G_12/13 signaling. The structural analysis identified F190^5.47 as a critical residue for curcumin binding. The importance of the methoxy group and heptadienone moiety in curcumin's activity was also highlighted. The study's findings support the idea that GPR55 mediates some of curcumin's physiological effects, particularly its anti-hyperglycemic action by stimulating GLP-1 secretion. Although curcumin's low bioavailability is a known limitation, the high curcumin concentrations achieved in the intestine suggest that GPR55 activation in the gut may contribute to its physiological effects. The fact that CID16020046 did not completely block GLP-1 secretion suggests the involvement of other receptors.

This study successfully identified GPR55 as a molecular target of curcumin, demonstrating its role as an agonist activating G_12/13 signaling. The identification of F190^5.47 as a crucial residue for curcumin binding and the confirmation of GPR55's involvement in GLP-1 secretion highlight the significance of this receptor in mediating curcumin's biological activities. Further research should focus on elucidating the full extent of GPR55's contribution to curcumin's multifaceted effects and exploring its potential therapeutic applications.

The study primarily focused on in vitro experiments. Further in vivo studies are needed to validate the findings in a whole-organism context. The study did not directly measure curcumin binding to GPR55, relying instead on functional assays and docking simulations. While the docking simulations and mutagenesis experiments provided strong evidence of interaction, a direct binding assay would strengthen the conclusions. The complete mechanism underlying curcumin's effects on GLP-1 secretion may involve other receptors in addition to GPR55.