Article Figures & Data
Figures
- Figure 1.
R9AP and GPR179 are colocalized in the majority of ON-BC synapses. A, Immunohistochemical analysis of R9AP and GPR179 colocalization with each other and with mGluR6 in the ON BC dendrites in OPL of mouse retinas. Triple staining with indicated antibodies was performed, and each channel is individually pseudo-colored. Arrows indicate the position of the cone synapses. Scale bar, 10 μm. B, Quantitative analysis of protein distribution across ON-BC synapses with rod and cone photoreceptors.
- Figure 2.
GPR179 is necessary for the correct localization of R9AP in retinal ON-BC synapses and the expression of RGS11 in the OPL. A, Immunohistochemical analysis of ON-BC synaptic components in the OPL of WT and Gpr179nob5 mouse retinas. Retina sections were immunostained with the indicated antibodies. Synaptic architecture in Gpr179nob5 retinas is intact as evidenced by normal apposition of presynaptic and postsynaptic markers (CtBP2, mGluR6, and TRPM1, respectively). Note the lack of accumulation of R9AP and complete absence of RGS11 in Gpr179nob5 ON-BC dendritic tips. Scale bar, 5 μm. B, Double immunolabeling of WT, Gpr179nob5, and RGS7/RGS11 DKO retinas with mGluR6 and R9AP antibodies. Scale bar, 5 μm. Quantification of the accumulation of R9AP within mGluR6-positive puncta of ON-BC dendrites in OPL (described in Materials and Methods). C, Corresponding values. Error bars indicate SEM. *p < 0.05 (one-way ANOVA with Bonferroni's post hoc test). **p < 0.01 (one-way ANOVA with Bonferroni's post hoc test). ***p < 0.001 (one-way ANOVA with Bonferroni's post hoc test). n = 25–30 puncta for each group. D, Top, High-magnification images of retina sections with scan-line analyses of R9AP fluorescence intensity across the midline of mGluR6-positive synaptic puncta in OPL. Bottom, Averaged traces of R9AP fluorescent intensity across the midline of mGluR6-positive synaptic puncta reveal a complete lack of after synaptic R9AP accumulation in Gpr179nob5 retinas, but only a partial loss in RGS7/11 DKO. n = 25–30 for all groups. Scale bar, 0.125 μm.
- Figure 3.
RGS and R9AP proteins are not required for the expression or correct localization of GPR179 in the retinal OPL. A, Immunohistochemical analysis of ON-BC synaptic components in the OPL of WT, R9AP, and RGS7/RGS11 DKO mouse retinas. Retinal sections were stained with the indicated antibodies. Scale bar, 5 μm. B, Double immunolabeling with mGluR6 and GPR179 antibodies. Scale bar, 5 μm. Quantification of the accumulation of GPR179 within mGluR6-positive synaptic puncta relative to the OPL (described in Materials and Methods). C, Corresponding values. Error bars indicate SEM; n = 30–35 puncta for each group. One-way ANOVA shows no significant differences. D, Top, High-magnification images of retinal sections with line-scan analyses of GPR179 fluorescence intensity across the midline of mGluR6-positive synaptic puncta in OPL. Bottom, Averaged traces of GPR179 fluorescent intensity across the midline of mGluR6-positive synaptic puncta reveal no changes to postsynaptic GPR179 accumulation in either R9AP KO or RGS7/RGS11 DKO retinas; n = 25–30 for all groups.
- Figure 4.
GPR179 and R9AP physically interact in vivo and in transfected cells. A, Immunoprecipitation of R9AP from retinas following detection of coeluting proteins by Western blotting. R9AP KO retinas were used as a negative control in a parallel experiment. GPR179 and RGS11 coimmunoprecipitate with R9AP from WT, but not from the R9AP KO retinas. B, Analysis of R9AP and GPR179 coimmunoprecipitation in transfected HEK293 cells. Top, Antibodies against R9AP are able to specifically coimmunoprecipitate GPR179. Bottom, Antibodies against GPR179 are able to coimmunoprecipitate R9AP.
- Figure 5.
Mapping of the binding determinants involved in the interactions between GPR179, R9AP, and RGS proteins. HEK293 cells were cotransfected with the indicated constructs, and coimmunoprecipitation assays were conducted. A, Schematic representation of GPR179 and R9AP organization with the annotation of the protein fragments deleted in the various constructs used in the binding experiments. B, Deletional mutagenesis of R9AP binding determinants in GPR179. An antibody against the myc-tagged deletion mutants of GPR179 was used for immunoprecipitation. R9AP was detected only after coimmunoprecipitation with GPR179-ΔCT2. C, D, Deletional mutagenesis of RGS11 and RGS7 binding determinants in GPR179, respectively. An antibody against the myc-tagged deletion mutants of GPR179 was used for immunoprecipitation. RGS11 (C) and RGS7 (D) were coimmunoprecipitated with GPR179-ΔCT2 and GPR179-ΔCT1 constructs. E, Deletional mutagenesis of GPR179 binding determinants in R9AP. An antibody against R9AP was used in the experiment. Deletion of the transmembrane region of R9AP does not affect its interaction with GPR179. F, Coimmunoprecipitation of R9AP and GPR179 deletional mutants containing minimal binding determinants. Asterisk indicates the position of nonspecific band detected in the immunoprecipitation eluates, which likely corresponds to IgGs used for affinity precipitation.
- Figure 6.
Elimination of RGS7 does not rescue loss of RGS11 expression and synaptic localization in R9AP knock-out retinas. A, Diagram model representing possible arrangement of RGS proteins and their membrane anchors GPR179 and R9AP at the dendritic tips of the ON-BC neurons. RGS7 is capable of binding only to GPR179, whereas RGS11 can bind to both GPR179 and R9AP. B, Western blot analysis of protein expression in retinas of RGS7/R9AP DKO mice in comparison with retinas lacking R9AP or RGS7 alone. There is no detectable RGS11 expression in either R9AP KO or R9AP/RGS7 DKO retinas. C, Analysis of synaptic targeting of proteins of interest in retinas lacking RGS7 and R9AP by immunohistochemistry. ON-BCs of R9AP/RGS7 DKO mice have normal cytoarchitecture and correct targeting of cascade components. There was no detectable accumulation of RGS11 at the synapses of ON-BCs in the R9AP/RGS7 DKO mice. Scale bar, 10 μm.
- Figure 7.
Concurrent knock-out of R9AP and RGS7 causes no b-wave (nob) ERG phenotype. A, Representative ERG traces from dark-adapted WT, RGS7 KO, R9AP KO, and RGS7/R9AP DKO mice retinas elicited with scotopic flashes. B, Representative ERG traces from dark-adapted WT, RGS7 KO, R9AP KO, and RGS7/R9AP DKO mice retinas elicited with photopic flashes. RGS7/R9AP DKO mice have a nob ERG phenotype in both scotopic and photopic ranges and a normal a-wave. C, Comparison of ERG light responses of RGS7/R9AP DKO mice with those recorded in TRPM1 KO mice under scotopic (left) and photopic (right) conditions. Overlay of the representative traces reveals no apparent differences.
- Figure 8.
Proposed model of the GAP complex configuration in ON-bipolar neurons. Cytosolic domains of GPR179 that comprise intracellular loops and coiled-coil containing RGS binding motif directly interact with SNARE-like intracellular domain of R9AP. R9AP binds exclusively to RGS11/Gβ5, and GPR179 binds exclusively to RGS7/Gβ5. Both protein complexes facilitate GTP hydrolysis on Gαo promoting its deactivation.
