Panoramic characterization of proposed ligands for GPR3: oleoylethanolamide as a pharmacological chaperone.

G protein-coupled receptor 3 (GPR3) is a constitutively active G protein-coupled receptor known to regulate β-amyloid deposition in the brain and metabolism in brown adipose tissue. Although multiple compounds have been proposed to activate GPR3 signaling, it is still considered an orphan receptor. Recently, structural evidence revealed a hydrophobic tunnel in GPR3, suggesting that a lipid may be an endogenous agonist for GPR3. We sought to characterize the reported agonists for GPR3 to determine which might be authentic agonists and found that only the synthetic ligand, diphenyleneiodonium chloride, and the endogenous lipids, oleoylethanolamide (OEA) and oleamide, stimulated cAMP generation in a GPR3-dependent manner. Although treatment of cells with diphenyleneiodonium chloride stimulated rapid cAMP generation, we observed gradual increases in GPR3 activation over multiple hours after OEA treatment. Additionally, we found that chronic stimulation with OEA caused an increase in GPR3 cell surface expression. Coupled with the slow activation kinetics of OEA-stimulated GPR3 signaling, these findings suggest that OEA does not behave strictly as a classical agonist for GPR3. Indeed, we found that the OEA-mediated increase in GPR3 on the cell surface can be blocked by inhibiting receptor forward trafficking through the endoplasmic reticulum and Golgi, which positions OEA as a pharmacological chaperone that increases GPR3 signaling by promoting receptor trafficking to the cell surface. These findings suggest a novel mechanism for the regulation of the constitutively active receptor GPR3, whereby the amount of receptor at the plasma membrane, and therefore, the receptor's signaling activity, can be regulated by lipid binding in the endoplasmic reticulum/Golgi. SIGNIFICANCE STATEMENT: This study proposes a novel mechanism for the regulation of the activity of the G protein-coupled receptor, G protein-coupled receptor 3. This study found that the endogenous lipid oleoylethanolamide functions as a pharmacological chaperone by promoting the accumulation of G protein-coupled receptor 3 at the plasma membrane through increased forward trafficking, thereby inducing increased receptor signaling activity.