Characterization of molecular forms of G protein-coupled receptor kinase 1 (rhodopsin kinase) in vertebrate retina and pineal gland
The purpose of my dissertation was to investigate the presence of molecular forms of G protein-coupled receptor kinase 1 (GRK1), and their functions in photoreceptor cells of the vertebrate retina and the pineal gland.GRK1 is a critical enzyme in photopigments inactivation and recovery of the photoreceptors to the dark state. Only one photoreceptor kinase, GRK1 was cloned from rod-dominant rat retina, cone-enriched human fovea, and cone-dominant chicken retina using a combination of molecular cloning and RT-PCR. In addition, GRK1 was localized to the outer segments of both human rods and cones by two monoclonal antibodies specific for human GRK1. Strong immunolabeling of chicken photoreceptor outer segments provided further evidence that GRK1 is present in both rods and cones. GRK1 is also expressed in mammalian and chicken pineal gland as demonstrated using molecular cloning methods. We have also cloned rod opsin and blue cone opsin, putative substrates of GRK1 from rat pineal, suggesting that both rod and cone pigments are substrates for GRK1. This is consistent with the fact that cone pigments are good substrates for GRK1 in vitro. The colocalization of opsin and GRK1 in human pinealocytes suggests a novel role of GRK1 in an extraocular phototransduction system. In searching for different isoforms of GRK1, we have cloned splice variants of human and chicken GRK1, GRK1b. Detailed characterization of human GRK1b indicates that GRK1b retained the last intron, intron 6. Although the mRNA of GRK1b is abundant and prevalent in humans and localized in photoreceptor cell cytosol, we only detected a low level of GRK1b protein in human retina, suggesting GRK1b is either unstable or is translated at low level. These data indicate the complex regulation of GRK1 at transcription and translational levels. In addition, a mutant form of GRK1 was found in a patient with Oguchi's disease. This patient was identified to have a deletion mutation in exon 5, which result in nonfunctional GRK1. The results obtained from psychophysical and electrophysiological studies indicate that GRK1 is a critical enzyme in the recovery of rod photoreceptor sensitivity, but play a minor role in the activation and adaptation to background illumination of photoreceptors. The small but definite delay of cone recovery in this Oguchi patient indicates that mechanisms other than phosphorylation by GRK1 may play predominant role in the inactivation of cone photoreceptors. Finally, biochemical data indicates that chicken GRK1 is likely to be geranylgeranylated, suggesting both geranylgeranylation and farnesylation of GRK1 are compatible with its function in the photoreceptor cells.
- Pharmacology