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.

Keywords: metabotropic glutamate receptor (mGluR); photoreceptor; retina; synapse; trafficking.

Figure 1

Deletion mutants.A, diagram of murine mGluR6 deletion mutants. Human CSNB missense mutations (36, 65, 66), with arrows indicating homologous positions in murine mGluR6, are shown. B, deleted regions shown in the structure of human mGluR4 (PDB 7E9H (67)) (homologous positions in mGluR4 are indicated in parentheses) in side views (top) and top views (LBD only, bottom). The mGluR6 region 112 to 137 has very low sequence similarity to mGluR4, and residues 128 to 147 in the homologous region are not visible in the mGluR4 structure (red arrows). The cytoplasmic CT domain is also absent in the mGluR4 structure. CRD, cysteine-rich domain; CT, C-terminal domain; LBD, ligand-binding domain; ss, signal sequence; TM, transmembrane domain.

Figure 2

Localization of mGluR6 mutants in WT retina.A, images of WT CD1 retinas electroporated with WT or mutant mGluR6-EGFP and immunostained with TRPM1 antibody. Boxes show location of higher magnification views on the right. B, quantification of mGluR6-EGFP OPL puncta, shown relative to total EGFP and normalized to WT. Dots represent means of at least three images each from biological replicates and error bars show means ± SEM. Quantification of endogenous mGluR6 labeled with antibody clone 366 (see Fig. 7) is included for comparison. C, quantification of total EGFP fluorescence in the same images used for (B). D, quantification of TRPM1 OPL puncta, shown relative to total TRPM1 staining and normalized to WT, in the same images used in (B). ^#p ≤ 0.001, one-way ANOVA with Dunnett’s post-test to compare all mutants to WT.

Figure 3

Localization of mGluR6 mutants in nob3 retina.A, images of CD1/nob3 retinas electroporated with WT or mutant mGluR6-EGFP and immunostained with TRPM1 antibody. Boxes show location of higher magnification views on the right. B–D, quantification of mGluR6-EGFP OPL puncta, total EGFP, and TRPM1 OPL puncta, as described in the Figure 2 legend. Dots represent biological replicates. ∗p ≤ 0.05; ∗∗p ≤ 0.01, one-way ANOVA with Dunnett’s posttest to compare all mutants to WT.

Figure 4

Reduced TRPM1 puncta accumulation in CD1/nob3 mice.A, retina sections from WT CD1 or CD1/nob3 mice were immunostained with antibodies for mGluR6 (clone 366), TRPM1, and ribeye. B, comparison of OPL puncta intensity in WT CD1 and CD1/nob3 retina sections. i, example quantification of technical replicate images from one experiment. Genotypes were compared with two-tailed unpaired t-tests. ii, means of biological replicates from three experiments; for each experiment, nob3 and WT retina sections were labeled on the same slide. Genotypes were compared with a two-tailed paired t test. C, magnified views from images as shown in (A). Brightness of the TRPM1 channel was increased for nob3 images to highlight the presence of residual TRPM1 at dendritic tips.

Figure 5

Rescue of TRPM1 dendritic tip localization by electroporated mGluR6-EGFP.A–E, i, examples of TRPM1 immunostaining in untransfected and transfected regions of the same retina. ii, example quantification of technical replicate images from one retina. Untransfected (untxf) and transfected (txf) regions were compared with two-tailed unpaired t-tests. iii, means of biological replicates were compared with two-tailed paired t-tests. F, summary of fold changes in TRPM1 puncta intensity in transfected regions. Asterisks on the bars indicate one-sample t test with theoretical mean of 1, and asterisks above the bars indicate comparison with WT using one-way ANOVA and Dunnett’s posttest. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001.

Figure 6

Intracellular location of mGluR6 LBD mutants. WT CD1 retinas electroporated with mGluR6-EGFP LBD mutants were immunostained for TRPM1 (A), ER marker BiP (B), or plasma membrane marker Na/K ATPase (C). Intensity profiles are shown for the lines overlaid on the images.

Figure 7

Specificity and fixation-dependent immunostaining of mGluR6 mAbs.A and B, WT C57 or nob3 eyes were fixed in 2% PFA for 10 min (A) or 4% PFA for 45 min (B), and sections were immunostained with an mGluR6 mAb, along with antibodies for TRPM1 and ribeye. C, western blots with WT C57 and nob3 retina tissue. The position of endogenous mouse IgG heavy chain dimers, detected with the secondary antibody, is indicated.

Figure 8

Intracellular location of somatic mGluR6. WT C57 retina sections were labeled with mGluR6 mAb 1438 and antibodies against TRPM1 (A), ER marker BiP (B), or plasma membrane marker Na/K ATPase (C). Intensity profiles are shown for the lines overlaid on the images.

Figure 9

mGluR6 mAb epitope mapping.A, diagram of mGluR6 with mAb epitopes shown. B and C, following preliminary mapping to segments N5, N7, and TM/CT as shown, western blots were performed with overlapping fragments fused to GST, and mGluR6 or GST antibodies. D, HEK cells were transfected with ss-Myc-mGluR6 and labeled in either permeabilizing or nonpermeabilizing conditions with both Myc and mGluR6 antibodies. E, structure of mGluR4 (PDB 7E9H) with positions homologous to mGluR6 mAb epitopes shown. Most of the CT epitope is not visible in the structure.

Figure 10

Surface expression and ELFN1 binding of mGluR6 mutants in heterologous cells.A and B, HEK293, HEK293F (shown), or CHO cells were transfected with mGluR6-EGFP and labeled in nonpermeabilizing conditions with mGluR6 mAb 1438 to detect surface expression. EGFP fluorescence in the same field was imaged to detect total expression. Image processing conditions that permitted visualization of WT mGluR6 1438 labeling resulted in oversaturated images for some mutants. Images processed independently are shown in Fig. S5B. Right, quantification of surface/total expression, shown normalized to WT. Points represent means from independent experiments. Inset, CT mutants shown on an expanded y-axis. C, extracellular domains of ELFN1 and LRRTM4 were fused to Fc, and secreted protein was bound to protein G beads. Bound beads were then incubated with lysate of HEK 293F cells transfected with mGluR6. Input lysate, flow-through lysate, and bead-bound samples were blotted with anti-human antibody and mGluR6 mAb 312. D, left, example dose–response curves showing glutamate-induced G-protein activation. Error bars: means ± SEM of technical replicates. Right, efficacy and pEC50 values. Points represent independent experiments, and error bars show means ± SEM. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ^#p ≤ 0.001, one-way ANOVA with Dunnett’s posttest to compare all mutants to WT.