Involvement of the C‐terminal domain in cell surface localization and G‐protein coupling of mGluR6

Abstract

Metabotropic glutamate receptor 6, mGluR6, interacts with scaffold proteins and Gβγ subunits via its intracellular C‐terminal domain (CTD). The mGluR6 pathway is critically involved in the retinal processing of visual signals. We herein investigated whether the CTD (residues 840–871) was necessary for mGluR6 cell surface localization and G‐protein coupling using mGluR6‐CTD mutants with immunocytochemistry, surface biotinylation assays, and electrophysiological approaches. We used 293T cells and primary hippocampal neurons as model systems. We examined C‐terminally truncated mGluR6 and showed that the removal of up to residue 858 did not affect surface localization or glutamate‐induced G‐protein‐mediated responses, whereas a 15‐amino acid deletion (Δ857‐871) impaired these functions. However, a 21‐amino acid deletion (Δ851‐871) restored surface localization and glutamate‐dependent responses, which were again attenuated when the entire CTD was removed. The sequence alignment of group III mGluRs showed conserved amino acids resembling an ER retention motif in the CTD. These results suggest that the intracellular CTD is required for the cell surface transportation and receptor function of mGluR6, whereas it may contain regulatory elements for intracellular trafficking and signaling.

The mGluR6 ligand-binding domain, but not the C-terminal domain, is required for synaptic localization in retinal ON-bipolar cells

“…A previous report examining CT deletion mutants of mGluR6 found that some mutants had reduced surface expression in heterologous cells, suggesting the presence of a regulatory element in this region ( 31 ). We also observed reduced absolute surface expression of the CT mutant Δ839–871, though total expression was also reduced.…”

Functional Optimization of Light-Activatable Opto-GPCRs: Illuminating the Importance of the Proximal C-terminus in G-protein Specificity

“…To investigate if the proximal C-terminus of mGluR6 is also involved in G-protein tropism, we next created an additional Mela(palm)-mGluR6(short CT) variant, where a truncated version of the mGluR6 C-terminus, commencing 3 AA downstream of the putative H8 considered devoid of G-protein interactions was employed (Rai et al, 2021) (Suppl. Fig.…”

Functional optimization of light-activatable Opto-GPCRs: Illuminating the importance of the proximal C-terminus in G-protein specificity

“…To investigate if the proximal C-terminus of mGluR6 is also involved in G-protein tropism, we next created an additional Mela(palm)-mGluR6(short CT) variant, where a truncated version of the mGluR6 C-terminus, commencing 3 AA downstream of the putative H8 considered devoid of G-protein interactions was employed (Rai et al, 2021) (Supplementary Figure S3). Indeed, Gi activation was significantly reduced (Figure 4C; p = 0.0009) in this short CT variant in favor of Gq activation when compared to the full CT Mela(palm) version, although the overall tropism of the chimera remained shifted to Gi (Figure 4A; 56.6% ± 0.2% Gi, 43.4% ± 0.04% Gq).…”