To determine whether mature cone photoreceptors express class I
A PI3K in vivo, we took advantage of the
Nrl −/− mouse model in which the photoreceptor population consists exclusively of cones by virtue of the absence of the rod differentiation transcription factor
Nrl.
48 Photoreceptor outer segments (POS) were isolated from WT and
Nrl −/− retinas and analyzed for p85α expression by Western blotting. Rod and cone photoreceptor-specific proteins rhodopsin and M-opsin were used as markers. The results indicate that mature cone POS express abundant amounts of p85α (
Fig. 1A) compared with POS from WT retinas. High levels of M-opsin were present in
Nrl −/− POS (
Fig. 1B) compared with WT POS. The rod-specific protein marker opsin was absent from
Nrl −/− POS lysates (
Fig. 1C), as previously established.
48 To further establish the existence of PI3K/Akt pathways in cone photoreceptors, we analyzed lysates of 661W cells (a cone-like transformed cell line
43 ) for p85α expression by Western blot analysis and immunofluorescence microscopy. This cell line has previously been used to study the multiple death pathways in cone photoreceptors.
49 Total retinal lysate from WT mice was used as a positive control. The results indicate that 661W cells express high levels of p85α protein (
Fig. 1D). Cone-like characteristics of 661W were confirmed by labeling 661W cell lysates (
Fig. 1E) or fixed 661W cells with anti–M-cone opsin antibody (
Fig. 1G). Further, the 661W cells do not express the rod photoreceptor marker rhodopsin (
Fig. 1F). The expression of p85α in 661W cells is shown in
Figure 1H. We found that the ratio of p85α expression to M-opsin was higher in 661W cells (
Figs. 1D,
1E) than in the POS
Nrl −/− mouse (
Figs. 1A,
1B). This difference was due to the whole cell lysate of 661W cell compared with POS in the
Nrl −/− mouse. The p85α in POS represents only the membrane-bound fraction, whereas in 661W cells it has both soluble and membrane-bound forms of p85α. These experiments provide evidence that cone photoreceptors express the p85α regulatory subunit of PI3K in vivo and in vitro.