Transducin β-Subunit Can Interact with Multiple G-Protein γ-Subunits to Enable Light Detection by Rod Photoreceptors

Abstract

The heterotrimeric G-protein transducin mediates visual signaling in vertebrate photoreceptor cells. Many aspects of the function of transducin were learned from knock-out mice lacking its individual subunits. Of particular interest is the knockout of its rod-specific γ-subunit (Gγ₁). Two studies using independently generated mice documented that this knockout results in a considerable >60-fold reduction in the light sensitivity of affected rods, but provided different interpretations of how the remaining α-subunit (Gα_t) mediates phototransduction without its cognate Gβ₁γ₁-subunit partner. One study found that the light sensitivity reduction matched a corresponding reduction in Gα_t content in the light-sensing rod outer segments and proposed that Gα_t activation is supported by remaining Gβ₁ associating with other Gγ subunits naturally expressed in photoreceptors. In contrast, the second study reported the same light sensitivity loss but a much lower, only approximately sixfold, reduction of Gα_t and proposed that the light responses of these rods do not require Gβγ at all. To resolve this controversy and elucidate the mechanism driving visual signaling in Gγ₁ knock-out rods, we analyzed both mouse lines side by side. We first determined that the outer segments of both mice have identical Gα_t content, which is reduced ∼65-fold from the wild-type (WT) level. We further demonstrated that the remaining Gβ₁ is present in a complex with endogenous Gγ₂ and Gγ₃ subunits and that these complexes exist in wild-type rods as well. Together, these results argue against the idea that Gα_t alone supports light responses of Gγ₁ knock-out rods and suggest that Gβ₁γ₁ is not unique in its ability to mediate vertebrate phototransduction.

Keywords: G-protein; phototransduction; retinal degeneration; transducin.

Graphical abstract

Figure 1.

Comparative analysis of Gα_t and Gβ₁ expression levels in retinal lysates from 1-month-old Gγ₁^−/− mice. A, Retinal lysates from Deltagen and StL mouse strains containing 10 μg of total protein were separated by SDS-PAGE alongside serial dilutions of WT retinal lysates and were immunoblotted with antibodies against each transducin subunit. Data are taken from one of three similar experiments. B, The relative contents of Gα_t and Gβ₁ in retinal lysates analyzed in A were determined from the calibration curves obtained from the WT lysate dilutions (Gα_t regression equation: y = 18,600 × x − 623; Gβ₁ regression equation: y = 8130 × x + 665). • = WT, ○ = Deltagen Gγ₁^−/−, and ♢ = StL Gγ₁^−/−. C, The amounts of transducin subunits in Gγ₁^−/− retinas averaged across three independent experiments are expressed as percentages of their amounts in WT retinas (mean ± SD). The amount of Gβ₁ was corrected to reflect that only 18% of Gβ₁ in Gγ₁^−/− retinas is expressed in rods, whereas the rest is expressed in the inner retina (Lobanova et al., 2008).

Figure 2.

Identification of alternative Gβ₁γ complexes in rod outer segments of Gγ₁^−/− and WT mice. A–D, Coimmunoprecipitation experiments were performed by incubating Gγ₁^−/− (A, B) or WT (C, D) rod outer segment lysates with mouse anti-Gγ₂ (A, C) or rabbit anti-Gγ₃ (B, D) antibodies. Immunoprecipitation with species-matched anti-β-actin (sc-47778) and anti-PSMD1 (ab140682) antibodies were used as negative controls; these antibodies were chosen based on the lack of cross-reactivity with the proteins analyzed in this panel, as evaluated in independent experiments. The data represent one of four similar experiments performed with Gγ₁^−/− or two similar experiments performed with WT outer segment preparations.

Figure 3.

Comparative analysis of retinal morphology in Deltagen and StL Gγ₁^−/− mice. A, Representative images of toluidine blue-stained, plastic-embedded 1-μm-thick retinal cross sections from each mouse strain at the indicated ages. Scale bar, 25 μm. B, The number of photoreceptor nuclei counted in 400 μm outer nuclear layer (ONL) segments located 1 mm from either side of the optic nerve. The number of nuclei in each segment was counted by hand. Data were averaged across nuclear counts obtained from each side of the optic nerve from three or four animals of each age and genotype, and are shown as the mean ± SD. Each dot represents a single data point. C, Accumulation of the Ub^G76V–GFP reporter of proteasomal activity (green) in rods of 3-month-old Deltagen and StL Gγ₁^−/− mice. A Ub^G76V–GFP-expressing WT retina is shown as a control. Wheat germ agglutinin staining is shown in red. Scale bar, 10 μm.