N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β2 adrenergic receptor (β2AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β2AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β2AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β2AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs.Enzymes
Peptide-N-glycosidase F (PNGase F, EC 18.104.22.168); endo-β-N-acetylglucosaminidase A (Endo-A, EC 22.214.171.124).
It remains unknown how N-glycosylation affects GPCR signaling. β2AR has three N-glycosylation sites: Asn6, Asn15, and Asn187. Our study described the role of N-glycosylation in β2AR expression and function. In addition, we put forward an idea that Asn6 and Asn15 glycosylation is critical for receptor homo-dimerization mediating efficient downstream signals.