R9AP stabilizes RGS11-G beta5 and accelerates the early light response of ON-bipolar cells

Abstract

The rate-limiting step in the recovery of the photoreceptor light response is the hydrolysis of GTP by transducin, a reaction that is accelerated by the RGS9-Gbeta5 complex, and its membrane anchor, R9AP. Similar complexes, including RGS7, RGS11, and Gbeta5, are found in retinal ON-bipolar cell dendrites. Here, we present evidence that R9AP is also expressed in the dendritic tips of ON-bipolar cells. Immunofluorescent staining for R9AP revealed a punctate pattern of labeling in the outer plexiform layer, where it colocalized with mGluR6. In photoreceptors, R9AP is required for proteolytic stability of the entire regulator of G protein signaling complex, and we found that genetic deletion of R9AP also results in a marked reduction in the levels of RGS11 and Gbeta5 in the bipolar cell dendrites; the level of RGS7 was unaffected, suggesting the presence of another interaction partner to stabilize RGS7. To determine the effect of R9AP deletion on the response kinetics of ON-bipolar cells, we compared the electroretinogram (ERG) between wild-type and R9AP-deficient mice. The ERG b-wave, reflecting ON-bipolar cell activity, was delayed and larger in the R9AP-deficient mice. Our data indicate that R9AP is required for stable expression of RGS11-Gbeta5 in ON-bipolar cell dendrites. Furthermore, they suggest that the RGS11-Gbeta5-R9AP complex accelerates the initial ON-bipolar cell response to light.

Figure 1. R9AP is localized to the tips of ON-bipolar cell dendrites

A. Vertical cryosections from wild-type (+/+) and R9AP deficient (−/−) mouse retina were labeled for R9AP by immunofluorescence. Left panel: Nomarsky image of the +/+ retina section in the middle panel. Abbreviations: OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. The scale bar represents 10 µm. B. Top row: Mouse retina section double labeled for R9AP (left, green) and PKCα (middle, red).Bottom row: OPL of a mouse retina section double labeled for R9AP (left, red) and mGluR6 (middle, green). Merged images are shown on the right; co-localization of the two antigens appears yellow. The scale bar applies to all panels and represents 10 µm.

Figure 2. The R9AP^−/− retina lacks RGS11 but has a normal OPL ultrastructure

A. Western blots of retinal extracts from R9AP +/+ and −/− littermates were probed for Gβ5, RGS7, RGS11 and R9AP. B. Retina sections from R9AP +/+ and −/− littermates were labeled by immunofluorescence for Gβ5, RGS7, and RGS11. C. Transmission electron micrograph showing normal ultrastructure of the OPL in the R9AP deficient mouse. The examples show a rod spherule (left) and a cone pedicle (right) showing normal invagination of postsynaptic horizontal cell processes (H) and bipolar cell dendrites (B) at the ribbon synapses (arrowheads). Scale bar = 0.2 µm. D. RGS11 western blot of: Lane 1: extract from cells transfected with RGS11, Gβ5, and R9AP precipitated with Protein G sepharose beads alone; Lane 2: extract immunoprecipitated with an antibody to R9AP and Protein G sepharose beads; Lane 3: total extract.

Figure 3. R9AP deficiency slows the ERG b-wave

A. Mean ERGs from WT (gray traces) and R9AP^−/− (black traces) mice for a representative range of flash intensities. Numbers to the left of traces indicate flash intensities in log sc cd-s/m². Note change of scale for flash intensities greater than 0 log sc cd-s/m². B. Mean ERGs to four high flash intensities shown over a shorter time base, highlight the similarity in the ERG a-waves between WT (gray traces) and R9AP^−/− (black traces) mice.

Figure 4. R9AP deficiency delays the onset of the rising phase of the ERG b-wave

A. ERG to a −2.5 log sc cd-s/m² flash (top). Filtered ERG b-wave (middle) was obtained following digital subtraction of the OP’s (bottom) from the ERG. B. Mean filtered ERG b-waves from WT (gray traces) and R9AP^−/− (black traces) mice to a −2.5 log sc cd-s/m² flash. Dotted lines show traces 1 S.E. either side of the mean. C. The means of the derivatives of the filtered ERGs to the −2.5 log sc cd-s/m² flash. The peak of the derivative (arrows) corresponds to the maximal slope of the rising phase of the filtered ERG b-wave. D. Latencies to reach maximum slope as a function of flash intensity. Error bars, ±SE

Figure 5. Isolation of the P2 response

A. ERG to a 1.8 log sc cd-s/m² flash (solid black line); Phototransduction P3 model fit to the leading edge of the ERG a-wave (blue line); Post-photoreceptoral ERG after subtraction of P3 model fit (dashed black line); P2 response following digital subtraction of OPs (red line) B. P2 responses from a WT mouse generated by flash intensities of 1.5, 1.8, 2.0 and 2.2 log sc cd-s/m² (responses to highest 3 flash intensities overlap).

Figure 6. R9AP deficiency delays the onset of the rising phase of the bright flash P2 response

For all graphs, gray traces are from WT mice and black traces are from R9AP^−/− mice. Dotted lines show traces 1 S.E. either side of the mean. A. Mean P2 responses to a 1.8 log sc cd-s/m² flash B. The means of the derivatives of the P2 response for a 1.8 log sc cd-s/m² flash. C. Latencies to reach maximum slope on the rising phase of the P2 response as a function of flash intensity. Error bars, ±SE