Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176

G-protein-coupled receptors (GPCRs) are important drug targets with diverse therapeutic applications. However, there are still more than a hundred orphan GPCRs, whose protein functions and biochemical features remain unidentified. Gpr176 encodes a class-A orphan GPCR that has a role in circadian clock regulation in mouse hypothalamus and is also implicated in human breast cancer transcriptional response. Here we show that Gpr176 is N -glycosylated. Peptide- N -glycosidase treatment of mouse hypothalamus extracts revealed that endogenous Gpr176 undergoes N -glycosylation. Using a heterologous expression system, we show that N -glycosylation occurs at four conserved asparagine residues in the N-terminal region of Gpr176. Deficient N -glycosylation due to mutation of these residues reduced the protein expression of Gpr176. At the molecular function level, Gpr176 has constitutive, agonist-independent activity that leads to reduced cAMP synthesis. Although deficient N -glycosylation did not compromise this intrinsic activity, the resultant reduction in protein expression was accompanied by attenuation of cAMP-repressive activity in the cells. We also demonstrate that human GPR176 is N -glycosylated. Importantly, missense variations in the conserved N -glycosylation sites of human GPR176 (rs1473415441; rs761894953) affected N -glycosylation and thereby attenuated protein expression and cAMP-repressive activity in the cells. We show that N -glycosylation is a prerequisite for the efficient protein expression of functional Gpr176/GPR176.