Abstract
Purpose: :
One subtype of Leber congenital amaurosis (LCA) is caused by a mutation in the gene encoding the aryl hydrocarbon receptor-interacting protein like 1 (AIPL1). Previous studies in mice have shown that rod cGMP phosphodiesterase (PDE6) holoenzyme, a critical phototransduction component, is concomitantly reduced in Aipl1 mutant retinas and that AIPL1 mutation leads to similar phenotypic changes as found in PDE6 mutant retinas. One PDE6 holoenzyme consists of 4 subunits (αβγ2). However, the mechanism underlying the AIPL1-PDE6 interaction is not fully understood. One aim of this study is to compare the retinal photoresponses between Aipl1 mutant mice and rd1 mice carrying a mutation in the β subunit of PDE6. The other aim is to reveal the AIPL1-PDE6 interacting mechanism by using cell biological and biochemical approaches.
Methods: :
Electroretinograms (ERG) for rod photoreceptors were recorded for both Aipl1-/- and rd1 mice before obvious loss of photoreceptor cells (P12). Co-immunoprecipitation (Co-IP), silver staining and Nano-HPLC/MS/MS were applied to study the AIPL1-PDE6 interaction.
Results: :
Both Aipl1-/- and rd1 mice showed severe reduction in amplitude in all elicited responses. In both groups, the severe deficits were evident in a-wave, b-wave and oscillatory potentials. Rod responses, standard combined responses, maximum combined responses in Aipl1-/- mice were significantly smaller in amplitude than in rd1 mice (p<0.009). It thus appears that the functional deficits in the Aipl1 mutant rods are more severe than those of the rd1 mice. Co-IP experiments found that the anti-AIPL1 antibody was able to pull down a 90-kDa protein. Sequence identification with Nano-HPLC/MS/MS indicated that this 90-kDa band contained α subunit of PDE6, which was also confirmed by immunoblotting. In addition, further binding assays demonstrated that the AIPL1-interacting motif is located in the C-terminal CAAX motif of α subunit, which is required for prenylation.
Conclusions: :
More severe rod functional deficits in the Aipl1 mutant retina than rd1 mice suggest that AIPL1 deficiency affects more than just the β subunit of the PDE6 holoenzyme. The interaction between AIPL1 and the CAAX motif of PDE6 suggests that AIPL1 may play a role in PDE6 prenylation or PDE6 prenylation may regulate the AIPL1-PDE6 interaction.
Keywords: chaperones • retinal degenerations: cell biology • photoreceptors