Targeting of RGS7/Gbeta5 to the dendritic tips of ON-bipolar cells is independent of its association with membrane anchor R7BP

Abstract

Complexes of regulator of G-protein signaling (RGS) proteins with G-protein beta5 (Gbeta5) subunits are essential components of signaling pathways that regulate the temporal characteristics of light-evoked responses in vertebrate retinal photoreceptors and ON-bipolar cells. Recent studies have found that RGS/Gbeta5 complexes bind to a new family of adapter proteins, R9AP (RGS9 anchor protein) and R7 family binding protein (R7BP), that in case of the RGS9/Gbeta5 complex were shown to determine its precise subcellular targeting to either the outer segment of photoreceptors or postsynaptic structures of striatal neurons, respectively. In this study, we establish that another trimeric complex consisting of RGS7, Gbeta5, and R7BP subunits is specifically targeted to the dendritic tips of retinal bipolar cells. However, examination of the mechanisms of complex targeting in vivo surprisingly revealed that the delivery of RGS7/Gbeta5 to the dendrites of ON-bipolar cells occurs independently of its association with R7BP. These findings provide a new mechanism for adapter-independent targeting of RGS/Gbeta5 complexes.

Figure 1.

Colocalization of RGS7 and R7BP in the retina. A, Localization of R7BP and RGS7 as determined by a serial sectioning approach. Distribution of R7BP and RGS7 in 10 μm sections from rat retina was compared with the localization of marker proteins: phosducin (Pdc) for photoreceptors and G-protein Gαo for bipolar cells. Colocalization of RGS9-1 and R9AP proteins in photoreceptors was used as a reference. B, Immunostaining of frozen retina cross sections with antibodies against RGS7 (green), R7BP (red), and bipolar cell marker PKCα (red). Merged images (yellow) reflect colocalization of proteins. Scale bar, 20 μm. C, High-magnification confocal images of the outer plexiform layer stained with the antibodies against RGS7 (red) and R7BP or mGluR6 (green). Merged channel (yellow) shows colocalization. Scale bar, 10 μm. The following abbreviations are used in this and subsequent figures: PR, photoreceptors; HC, horizontal cells; BC, bipolar cells, AC, amacrine cells; GC, ganglion cells; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; OPL, outer plexiform layer; IPL, inner plexiform layer.

Figure 2.

Protein expression in mouse knock-outs lacking components of RGS complexes. A, Analysis of morphology in plastic sections reveals no difference between wild-type (WT), R7BP knock-out (R7BP KO), or RGS11 knock-out (RGS11 KO) retinas. Scale bar, 25 μm. B, Western blot analysis of protein expression in the retinas of wild-type and R7BP KO mice; 8.5 μg of total retina protein was loaded in each lane. C, Analysis of protein levels in retinas of mice lacking components of R7 RGS protein complexes by Western blotting.

Figure 3.

Localization of RGS7/Gβ5 in R7BP knock-out retinas. A, Subcellular fractionation shows shift of RGS7 to cytoplasmic fraction in R7BP knock-out retinas. Retina lysates were separated into soluble cytoplasmic (S) and insoluble membrane (P) compartments by centrifugation. The presence of proteins in each fraction was revealed by Western blotting. B, Quantification of data presented in A from three independent experiments. RGS7 band intensities were determined by densitometry using ImageJ software (NIH) and plotted as percentage of total density. Error bars indicate SEM. C, Analysis of RGS7 and Gβ5 localization in retina cross sections by immunostaining. Sections from Gβ5 and R7BP knock-outs were used as a control for nonspecific staining. Scale bar, 20 μm. D, High-power images of RGS7 immunoreactivity (green) in synapses of ON-bipolar cells stained with anti-PKCα antibodies (red). Distinct green puncta correspond to RGS7 enriched in dendritic tips of bipolar cells. Scale bar, 10 μm. KO, Knock-out; WT, wild type.

Figure 4.

Light-evoked responses of R7BP KO retinas. A–D, Electrophysiological responses were analyzed by ERG (A) and single-cell voltage-clamp recordings from ON-bipolar cells (B–D). A, ERGs from wild type (R7BP+/+) and R7BP−/− mice. Shown are typical responses evoked by −40, −30, −20, and −10 dB UTAS-E400 flashes corresponding to the indicated illuminance in candelas per second per square meter. Traces were obtained using 300 Hz high-cut filter setting and the averaging of 5–10 records. B, Light-evoked inward currents in rod bipolar cells were plotted as a function of time for brief flashes (arrowheads) of 0.52, 1.2, 2.7, 5.4, 11, and 22 Rh*/rod. The bandwidth was 50 Hz. C, Estimated responses per photon plotted as a percentage of maximum inward current. The R7BP+/+ response (solid) was the average of 244 responses across 14 rod bipolar cells resulting from a flash that on average generated 0.9 Rh*/rod. The R7BP−/− response (dotted) was the average of 211 responses across 16 rod bipolar cells resulting from a flash that on average also generated 0.9 Rh*/rod. D, Response–intensity relationship across all rod bipolar cells in R7BP+/+ (n = 14) and R7BP−/− (n = 16) mice. Data are plotted as mean ± SD, and the least-squares Hill curve fits to the mean and SDs are superimposed. For R7BP+/+ rod bipolar cells, I_1/2 = 2.7 Rh*/rod, and n = 1.56 (solid). For R7BP−/− rod bipolar cells, I_1/2 = 2.7 Rh*/rod, and n = 1.64 (dotted).