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.
Highlights
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
Deletion mutations within the ligand-binding domain (LBD) of mGluR6 revealed that multiple regions, or a tertiary structure requiring multiple regions, are required for dendritic tip localization in bipolar cells (BCs) (Fig. 2)
All LBD mutants were unable to bind to ELFN1 (Fig. 10), raising the possibility that ELFN1 binding is linked to dendritic tip localization
Summary
The mGluR6 ligand-binding domain, but not the C-terminal tail, is necessary for dendritic tip localization in ON-BCs. LBD mutants had drastically reduced OPL puncta intensity (Fig. 2B), while TRPM1 was similar for all mutants, showing that dendritic tips were. Similar to the mislocalized LBD mutants, endogenous somatic mGluR6 detected with clone 1438 was partially colocalized with TRPM1 and BiP, but not the plasma membrane marker Na/K ATPase (Fig. 8). All constructs had similar labeling with 1438 (Fig. S5A), indicating that the enhanced surface expression seen with some mutants was not due to different accessibility of the mAb epitope. Protein-G beads with bound Fc fusions were incubated with detergent-solubilized lysates of HEK cells expressing untagged mGluR6 In this assay, WT mGluR6, but none of the LBD mutants, bound to ELFN1 (Fig. 10C). Since Δ839–871 was correctly localized to dendritic tips in BCs (Figs. 2 and 3), these results indicate that functional G-protein activation is not required for localization
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
