Functional redundancy of R7 RGS proteins in ON-bipolar cell dendrites

Abstract

Purpose: In the Gbeta5(-/-) mouse, the electroretinogram (ERG) b-wave is absent, and the R7 subfamily of regulators of G protein signaling (RGS), which includes RGS6, -7, -9, and -11, is downregulated. Mutant mouse strains deficient in RGS7 or -11 were characterized, and the SG711 strain which is deficient in both proteins was examined, to learn whether the loss of some of these RGS proteins causes the absence of the ERG b-wave.

Methods: Antibodies to RGS7 and -11 were generated to determine their expression levels and localizations in retinas with various genetic backgrounds by Western blot analysis and immunohistochemistry, respectively. The implicit times and amplitudes of ERG a- and b-waves were analyzed to examine photoreceptor and bipolar cell functions.

Results: RGS7 and -11 co-localized to the dendritic tips of the ON-bipolar cells. In the RGS11(-/-) mouse, the level of RGS7 protein increased. However, the level of RGS11 protein remained unchanged in the RGS7 mutant mouse, where a truncated RGS7 protein was expressed due to the deletion of exon 10. In the SG711 mouse retina, the Gbeta5-S protein level was reduced. The ERG b-wave of SG711 mice was markedly delayed. In contrast, RGS11(-/-) mice showed a moderately delayed b-wave, whereas the RGS7 mutant mice showed normal ERG responses.

Conclusions: The data demonstrate the presence of a delayed ERG b-wave in SG711 mice and a functionally redundant role for RGS11 and -7 at the tips of ON-bipolar cell dendrites. These results suggest that RGS11 or -7 works as the major physiological GAP (GTPase acceleration protein) for Galphao1 in ON-bipolar cells.

Figure 1.

The level of R7 RGS proteins in retinas of various genetically modified mouse lines. (A) Western blot analysis of retinal extracts derived from wild-type (lane 1) and Gβ5^−/− (lane 2) mice probed with the following antibodies: CT215 (anti-Gβ5), VCU008 (anti-RGS11), 7RC-1 (anti-RGS7), and IMG554 (anti-RGS6). (B) Western blot analysis showing the level of three R7 RGS proteins in retinal extracts derived from wild-type (lane 1), SG7 (lane 2), RGS11^−/− (lane 3), and SG711 (lane 4) mice. Arrows: GAPDH signal used as an internal loading control.

Figure 2.

Co-localization of RGS7 and -11 at the tips of DBC dendrites. A maximum projection transformation of a 3.2-μm z-stacked confocal image of the OPL of a GGFP5 mouse retinal section is shown. The retinal section was stained with two rabbit polyclonal antibodies, H190 (anti-RGS7) and VCU008 (anti-RGS11), in conjunction with the rabbit IgG labeling kit. Most cone terminals in the GGFP5 retina are visible under direct EGFP fluorescence. Both RGS7 and -11 are expressed in dendritic tips of DBCs and virtually all tips express both proteins. Scale bar, 5 μm.

Figure 3.

Upregulation of RGS7 level in RGS11^−/− mouse retinas. (A) A representative immunoblot showing RGS7, Gβ5 spliced forms, and GAPDH levels in wild-type (WT) and RGS11^−/− mouse retinas. (B) Quantification of RGS7 signal strength as normalized to the level of GAPDH between the two genotypes. The levels of Gβ5-L and -S are similar (data not shown) but a significant increase in RGS7 level is apparent (n = 6).

Figure 4.

Exon10 deletion of RGS7 reduced both the size and the level of RGS7 in SG7 mouse retinas. (A) A representative immunoblot showing the level of the truncated mutant RGS7 protein in SG7 versus WT mouse retinas. (B) Quantification of the level of reduction in RGS7 using GAPDH signal as a normalization factor (n = 8). A significant 50% decrease of overall RGS7 level was found in the SG7 retinas, whereas the level of RGS11 remained unchanged (data not shown).

Figure 5.

Downregulation of Gβ5-S in SG711 mouse retinas. (A) Representative immunoblots showing the level of Gβ5-S and -L in serially diluted retinal extracts (10, 5, and 2.5 μg protein/well from high to low) derived from WT and SG711 mice. (B) Quantification of Gβ5-S versus Gβ5-L signals between WT and SG711 mice (n = 8). A significant ∼30% decrease in Gβ5-S level is found in SG711 mice.

Figure 6.

Delayed ERG b-wave in RGS11^−/− and SG711 mice. Representative scotopic ERG responses from the four genotypes are shown. Flash intensity is indicated at left (in log cd s m⁻²).

Figure 7.

Measurement of ERG b-wave implicit time (A) and amplitude (B) as a function of flash intensity in mice of different genotypes. WT: n = 15; SG7: n = 8; RGS11^−/−: n = 13; and SG711: n = 10. No difference between WT and SG7 responses was found, although significantly larger b-wave implicit time (the time between flash onset and the peak of the b-wave) was present in the SG711 and RGS11^−/− mice, compared with the WT control mice.