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

Abstract

Signals from retinal photoreceptors are processed in two parallel channels—the ON channel responds to light increments, while the OFF channel responds to light decrements. The ON pathway is mediated by ON type bipolar cells (BCs), which receive glutamatergic synaptic input from photoreceptors via a G-protein-coupled receptor signaling cascade. The metabotropic glutamate receptor mGluR6 is located at the dendritic tips of all ON-BCs and is required for synaptic transmission. Thus, it is critically important for delivery of information from photoreceptors into the ON pathway. In addition to detecting glutamate, mGluR6 participates in interactions with other postsynaptic proteins, as well as trans-synaptic interactions with presynaptic ELFN proteins. Mechanisms of mGluR6 synaptic targeting and functional interaction with other synaptic proteins are unknown. Here, we show that multiple regions in the mGluR6 ligand-binding domain are necessary for both synaptic localization in BCs and ELFN1 binding in vitro . However, these regions were not required for plasma membrane localization in heterologous cells, indicating that secretory trafficking and synaptic localization are controlled by different mechanisms. In contrast, the mGluR6 C-terminus was dispensable for synaptic localization. In mGluR6 null mice, localization of the postsynaptic channel protein TRPM1 was compromised. Introducing WT mGluR6 rescued TRPM1 localization, while a C-terminal deletion mutant had significantly reduced rescue ability. We propose a model in which trans-synaptic ELFN1 binding is necessary for mGluR6 postsynaptic localization, whereas the C-terminus has a role in mediating TRPM1 trafficking. These findings reveal different sequence determinants of the multifunctional roles of mGluR6 in ON-BCs.

Complex N-glycosylation of mGluR6 is required for trans-synaptic interaction with ELFN adhesion proteins

“…7 ). WT mGluR6-EGFP exhibited normal dendritic tip localization, as we reported previously ( 18 , 32 ). All single mutants were capable of enrichment in TRPM1-containing puncta in the OPL ( Fig.…”

“…The predicted molecular mass of the mGluR6 precursor protein is ∼95 kDa and that of the mature protein after cleavage of the N-terminal signal peptide is ∼92 kDa. In SDS-PAGE, mGluR6 from rodent retina tissue migrates as a presumptive dimer of ∼200 kDa ( 2 , 18 , 23 , 24 ). We observed that mouse mGluR6 expressed in HEK cells by transient transfection migrated as a doublet near the expected dimer size.…”

“…Immunolabeling in nonpermeabilizing conditions with an extracellular epitope antibody (mAb-1438) ( 18 ) was used to assay PM localization in HEK cells ( Fig. 5 , A and C ).…”

“…mGluR6 is a class C GPCR with a large extracellular domain (ECD) that comprises a Venus flytrap ligand-binding domain (LBD) and a cysteine-rich domain (CRD) ( 19 ). Previously, we found that regions in the LBD were necessary for both ELFN1 binding and dendritic tip localization in BCs, but not plasma membrane (PM) trafficking in heterologous cells ( 18 ). The small C-terminal cytoplasmic domain was dispensable for dendritic tip localization in BCs, though deletions in this domain were reported to affect PM trafficking ( 18 , 20 , 21 ).…”

“…The mechanisms that target mGluR6 to postsynapses in BCs are unknown, though it has been suggested that trans-synaptic interactions with ELFN proteins may be required to recruit and/or maintain mGluR6 at the synapse ( 6 , 7 , 18 ). mGluR6 is a class C GPCR with a large extracellular domain (ECD) that comprises a Venus flytrap ligand-binding domain (LBD) and a cysteine-rich domain (CRD) ( 19 ).…”