N-Glycosylation of mGluR6 Modulates Receptor Cell-Surface Transport, G-Protein Coupling, and Interactions With Synaptic Adhesion Molecules.

Metabotropic glutamate receptor 6 (mGluR6) is a synaptic receptor expressed predominantly in retinal ON-bipolar cells. The N-terminal extracellular domain (ECD) of mGluR6 engages in ligand binding and also participates in mGluR6 cell-surface localization and trans-synaptic cell-adhesion complex formation. We herein investigated whether N-glycosylation in the mGluR6 ECD is involved in modulating receptor cell-surface transport, G-protein coupling, and interactions with Elfn1 and Lrit1 using 293T cells expressing ECD mutants with the asparagine-to-glutamine (N-to-Q) substitution in N-glycosylation sequons. The results obtained showed that mGluR6 underwent N-glycosylation at the asparagine residues N290, N445, N473, and N561 in the ECD, and the simultaneous blockage of N-glycosylation at these sites reduced cellular mGluR6 levels. Furthermore, mGluR6 cell-surface levels were decreased by N290Q and N445Q, but unaffected by N473Q and N561Q, while each N-to-Q substitution at the respective sites consistently impaired glutamate-induced G-protein-mediated responses. We also showed that mGluR6 was pulled down with Elfn1 in vitro, and coimmunoprecipitated with Lrit1 in co-transfected cells. The mGluR6-Elfn1 interaction was inhibited by N445Q and facilitated by N473Q and N561Q, whereas the mGluR6-Lrit1 interaction was promoted by N290Q and suppressed by N561Q. Collectively, these results indicate that each of the N-glycosylation sites contributed to efficient G-protein coupling, while the respective sites were involved in the modulation of receptor cell-surface transport and adhesion complex formation with Elfn1 and also with Lrit1 in distinct directions. Therefore, the N-glycosylation of synaptic receptors may have roles in modulating trans-synaptic bridge formation, potentially influencing the tuning of signal transmission.