Abstract
Purpose :
Timely deactivation of photoexcited visual pigment is essential for normal vision and failing to do so causes night blindness and photoreceptor degeneration. The G protein-coupled receptor kinase 1 (GRK1) is the enzyme responsible for phosphorylating visual pigments in rod and cone and its loss-of-function mutations are found in many human Oguchi disease patients. It is widely thought that truncated or mutant GRK1 loses its catalytic activity toward visual pigments, however, we present here a novel disease-causing mechanism in that the V380D mutation of GRK1 leads to protein instability in photoreceptors.
Methods :
We generated a transgenic mouse line where expression of a V380D mutant bovine Grk1 cDNA is put under the control of a mouse rhodopsin promoter. We mated the mice into the wild type (WT) and Grk1 knockout (-/-) backgrounds. We also generated a V380D knock-in mouse line by CRISPR/Cas9-based gene editing. Phospho-specific GRK1 antibody recognizing its autophosphorylation sites was made and validated, as well as several phospho-specific rhodopsin antibodies recognizing specific phosphorylated Serine residues at its C-terminal tail. Immunohistochemistry was then used to characterize the catalytic activity of WT and V380D mutant GRK1 in different genetic backgrounds using these phospho-specific antibodies.
Results :
Transgenic overexpression of the mutant Grk1 is readily detectable by RT-PCR, but it does not elevate the total GRK1 level judged by Western Blot. When mated to the Grk1 knockout background, transgenic V380D GRK1 protein becomes detectable at a ~20% WT level. Surprisingly, the transgenic V380D GRK1 retains some catalytic activity toward its own authophosphorylation sites and toward rhodopsin’s C-terminal substrate sites. On the contrary, the mutant V380D protein is hardly detected in the homozygous V380D knock-in mouse retinas and as a result, light-dependent rhodopsin phosphorylation does not occur, mimicking the pathophysiology of human Oguchi disease by failing to deactivate photoexcited rhodopsin in a timely manner.
Conclusions :
Our data suggests that protein instability, rather than a lack of catalytic activity, is the disease-causing mechanism underlying human Oguchi disease involving the Grk1 V380D mutations.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.