OPSINS WITH MUTATIONS AT THE SITE OF CHROMOPHORE ATTACHMENT CONSTITUTIVELY ACTIVATE TRANSDUCIN BUT ARE NOT PHOSPHORYLATED BY RHODOPSIN KINASE

More than 70 mutations in the gene encoding the visual pigment rhodops in have been identified in patients with autosomal dominant retinitis pigmentosa. Most of these mutations are thought to interfere with prop er folding of the membrane protein. However, families with a severe ph enotype of retinitis pigmentosa have been identified and shown to carr y a mutation at the site of chromophore attachment, Lys-296. This muta tion disrupts the inactive conformation of opsin and results in a cons titutively active protein that can activate the rod-specific GTP-bindi ng protein, transducin, in the absence of light and in the absence of the chromophore ll-cis-retinal. It has been suggested that this mutant opsin molecule may cause rod degeneration by depletion of the compone nts used to inactivate rhodopsin, such as rhodopsin kinase. In this wo rk we test this idea by determining whether two constitutively active opsin mutants are phosphorylated by rhodopsin kinase. We found that op sin mutants where Lys-296 is replaced either by Glu (K296E) or by Gly (K296G) are not substrates of rhodopsin kinase in the absence of chrom ophore. However, when K296G is regenerated with a Schiff base complex of ll-cis-retinal and n-propylamine and exposed to illumination, phosp horylation of opsin occurs. These experiments suggest that in the rod photoreceptors of patients with retinitis pigmentosa carrying a mutati on at Lys-296, there is persistent activation of the GTP-binding prote in-mediated cascade. This may result in a situation that mimics long-t erm exposure to continuous illumination and results in the degeneratio n of photoreceptors.