Abstract
Purpose: :
Rod PDE6, a key enzyme in the vertebrate visual signaling process is a heterotetrameric protein complex consisting of catalytic subunits, α and β and two inhibitory subunits (γ). Among all known proteins, rod PDE6 is unique in that it is the only multimeric protein that undergoes differential lipidation. The α-subunit is modified by a C-15 farnesyl lipid group. In contrast, β-subunit is linked to a more hydrophobic C-20 geranylgeranyl group. The reason behind variation in this highly conserved lipid modification and the role it plays in the functioning of PDE6 is not known and is the focus of this study.
Methods: :
We generated transgenic mice that express either wild-type PDE6 β with geranylgeranylated C-termini or a mutant PDE6 β subunit that will be farnesylated. Both wild-type and mutant alleles of PDE6 β were expressed under 4.4 kb rod opsin promoter with haemagglutinin (HA) tag at the N terminus. Transgenic positive mice were backcrossed with rd/rd (PDE6 β mutant) mice. Transgenic expression was checked by immunoblotting using antibodies against HA tag or PDE6 β subunit. Rod photoreceptor function was evaluated by electroretinography (ERG).
Results: :
The transgenic mice expressing wild-type PDE6 β shows robust rescue of photoresponse in rd/rd mice at P30, P60, P90 and P150 days. A similar response was observed in transgenic mice expressing the PDE6 β mutant allele at early stages (P30). However, this response is gradually lost by P150. This result suggests a slow and progressive photoreceptor specific degeneration due to the lack of proper lipid modification of PDE6 β subunit. We are currently undertaking morphological and biochemical characterization of these animal models to pinpoint the mechanism behind this photoreceptor degeneration.
Conclusions: :
Our study demonstrates the importance of differential lipid modification or geranylgeranylation of PDE6 β in proper functioning of rod photoreceptors. Defects in lipid modification also affect the survival of rod cells. Future studies will reveal the need for this differential lipid modification in functioning of PDE6.
Keywords: photoreceptors • retinal degenerations: cell biology • electroretinography: non-clinical