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C. W. Morgans, T. Puthussery, R. L. Brown, T. G. Wensel, B. Jeffrey, R. M. Duvoisin; Gβ5-RGS Complexes Interact With mGluR6 in Retinal On-Bipolar Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1285. doi: https://doi.org/.
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Photoexcitation of retinal photoreceptors decreases the rate of glutamate release from synaptic terminals, and the resulting deactivation of dendritic mGluR6 receptors causes of ON-bipolar cells (ON-BPCs) to depolarize in response to light. Little is known about this intracellular signaling pathway, however, beyond the fact that mGluR6 signals via a G-protein (Go) to open an as-yet-unidentified cation channel. We have recently identified complexes between the G-protein β subunit, Gβ5, and regulator of G-protein signaling (RGS) proteins, RGS7 and RGS11, in ON-BPC dendrites, and we are investigating the role of these complexes in the mGluR6 signal transduction pathway.
For immunohistochemistry, eye cups were fixed for 15-20 min in 4% paraformaldehyde. Slide-mounted cryosections (15 µm) were incubated for 1 - 2 h with approprite dilutions of primary and fluorescently-tagged secondary antibodies. Fluorescent staining was visualized by confocal microscopy. In other experiments, proteins were immunoprecipitated from retinal extracts with antibodies against Gβ5 or RGS proteins. Retinal extracts and immunoprecipitations were separated by SDS-PAGE, and specific proteins detected by immunoblotting.
We have found that both RGS7-Gβ5 and RGS11-Gβ5 are dependent on mGluR6 for proper localization to ON-BPC dendrites. In the nob4 mouse, a point mutation causes a total loss of the mGluR6 receptor protein. In the nob4 retina, we find that immunostaining for the RGS-Gβ5 complexes is dramatically reduced in the OPL, and instead, faint staining appears throughout the ON-BPCs. As determined by immunoblotting, however, absolute protein levels of Gβ5, RGS7, and RGS11 are increased in the nob4 retina compared to wild-type. Furthermore, we have discovered a differential dependence of RGS7-Gβ5 and RGS11-Gβ5 on the membrane anchor protein, R9AP. In R9AP-/- mice, RGS11 is undetectable in the retina by either immunoblotting or immunohistochemistry, whereas the level of RGS7 appears elevated.
Our data suggest that mGluR6 is part of a macromolecular signaling complex that also contains RGS7-Gβ5, RGS11-Gβ5, and R9AP. Our data further suggest that mGluR6 is required for the stability and localization of this complex.
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