The cloning of GRK7, a candidate cone opsin kinase, from cone- and rod-dominant mammalian retinas.
OBJECTIVE
Desensitization in the rod cell of the mammalian retina is initiated when light-activated rhodopsin is phosphorylated by the G protein-coupled receptor kinase (GRK), GRK1, often referred to as rhodopsin kinase. A distinct kinase that specifically phosphorylates cone opsins in a similar manner has not been identified in mammals. To determine the existence of a cone opsin kinase, RNA from the retinas of cone- and rod-dominant mammals was analyzed by PCR.


METHODS
RNA prepared from the retinas of two cone-dominant mammals, the thirteen-lined ground squirrel and the eastern chipmunk, and a rod-dominant mammal, the pig, was used to clone a new GRK family member by RT-PCR. The tissue distribution and localization of the kinase in retina were determined by Northern blot hybridization and in situ hybridization. The protein encoded by this cDNA was expressed in human embryonic kidney-293 (HEK-293) cells and compared with bovine GRK1 for its ability to phosphorylate bovine rhodopsin and to undergo autophosphorylation.


RESULTS
The cDNA cloned from ground squirrel contains an open reading frame encoding a 548 amino-acid protein. Sequence analysis indicates that this protein is orthologous to GRK7 recently cloned from O. latipes, the medaka fish. Partial cDNA fragments of GRK7 were also cloned from RNA prepared from eastern chipmunk and pig retinas. In situ hybridization demonstrated widespread labeling in the photoreceptor layer of the ground squirrel retina, consistent with expression in cones. Recombinant ground squirrel GRK7 phosphorylates bovine rhodopsin in a light-dependent manner and can be autophosphorylated, similar to bovine GRK1.


CONCLUSIONS
These results indicate that cone- and rod-dominant mammals both express GRK7. The presence of this kinase in cones in the ground squirrel and its ability to phosphorylate rhodopsin suggests that it could function in cone cells as a cone opsin kinase.
Weiss
E R
ER
Department of Cell Biology and Anatomy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, USA.
Raman
D
D
Shirakawa
S
S
Ducceschi
M H
MH
Bertram
P T
PT
Wong
F
F
Kraft
T W
TW
Osawa
S
S
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005399:Fishes / Q000235:genetics; D054768:G-Protein-Coupled Receptor Kinases; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D012313:RNA / Q000032:analysis; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D012160:Retina / Q000737:chemistry*; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry*; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000378:metabolism; D012589:Sciuridae / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine / Q000235:genetics; D014018:Tissue Distribution
D012313:RNA; D012243:Rhodopsin; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C111571:GRK7 protein, human; D054768:G-Protein-Coupled Receptor Kinases