GRK1-dependent phosphorylation of S and M opsins and their binding to cone arrestin during cone phototransduction in the mouse retina.
The shutoff mechanisms of the rod visual transduction cascade involve G-protein-coupled receptor (GPCR) kinase 1 (GRK1) phosphorylation of light-activated rhodopsin (R*) followed by rod arrestin binding. Deactivation of the cone phototransduction cascade in the mammalian retina is delineated poorly. In this study we sought to explore the potential mechanisms underlying the quenching of the phototransduction cascade in cone photoreceptors by using mouse models lacking rods and/or GRK1. Using the "pure-cone" retinas of the neural retina leucine zipper (Nrl) knock-out (KO, -/-) mice (Mears et al., 2001), we have demonstrated the light-dependent, multi-site phosphorylation of both S and M cone opsins by in situ phosphorylation and isoelectric focusing. Immunoprecipitation with affinity-purified polyclonal antibodies against either mouse cone arrestin (mCAR) or mouse S and M cone opsins revealed specific binding of mCAR to light-activated, phosphorylated cone opsins. To elucidate the potential role of GRK1 in cone opsin phosphorylation, we created Nrl and Grk1 double knock-out (Nrl-/-Grk1-/-) mice by crossing the Nrl-/- mice with Grk1-/- mice (Chen et al., 1999). We found that, in the retina of these mice, the light-activated cone opsins were neither phosphorylated nor bound with mCAR. Our results demonstrate, for the first time in a mammalian species, that cone opsins are phosphorylated and that CAR binds to phosphorylated cone opsins after light activation.
Zhu
Xuemei
X
The Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, Department of Cell and Neurobiology, the Keck School of Medicine of the University of Southern California, Los Angeles, California 90089-9112, USA.
Brown
Bruce
B
Li
Aimin
A
Mears
Alan J
AJ
Swaroop
Anand
A
Craft
Cheryl M
CM
D000818:Animals; D019393:Arrestin / Q000737:chemistry / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors; D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics; D003624:Darkness; D005136:Eye Proteins / Q000235:genetics; D051606:G-Protein-Coupled Receptor Kinase 1; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008027:Light; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D011494:Protein Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000502:physiology*; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D017299:Rod Opsins / Q000737:chemistry / Q000378:metabolism*; D014785:Vision, Ocular / Q000502:physiology
D019393:Arrestin; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D005136:Eye Proteins; C084884:Nrl protein, mouse; D017299:Rod Opsins; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; C496762:Grk1 protein, mouse