title: Effect of g protein-coupled receptor kinase 1 (Grk1) overexpression on rod photoreceptor cell viability.
abstract: OBJECTIVE
  Photoreceptor rhodopsin kinase (Rk, G protein-dependent receptor kinase 1 [Grk1]) phosphorylates light-activated opsins and channels them into an inactive complex with visual arrestins. Grk1 deficiency leads to human retinopathy and heightened susceptibility to light-induced photoreceptor cell death in the mouse. The goal of this study was to determine whether excess Grk1 activity is protective against photoreceptor cell death.
  
  
  METHODS
  Grk1-overexpressing transgenic mice (Grk1(+)) were generated by using a bacterial artificial chromosome (BAC) construct containing mouse Grk1, along with its flanking sequences. Quantitative reverse transcription-PCR, immunoblot analysis, immunostaining, and activity assays were combined with electrophysiology and morphometric analysis, to evaluate Grk1 overexpression and its effect on physiologic and morphologic retinal integrity. Morphometry and nucleosome release assays measured differences in resistance to photoreceptor cell loss between control and transgenic mice exposed to intense light.
  
  
  RESULTS
  Compared with control animals, the Grk1(+) transgenic line had approximately a threefold increase in Grk1 transcript and immunoreactive protein. Phosphorylated opsin immunochemical staining and in vitro phosphorylation assays confirmed proportionately higher Grk1 enzyme activity. Grk1(+) mice retained normal rod function, normal retinal appearance, and lacked evidence of spontaneous apoptosis when reared in cyclic light. In intense light, Grk1(+) mice showed photoreceptor damage, and their susceptibility was more pronounced than that of control mice with prolonged exposure times.
  
  
  CONCLUSIONS
  Enhancing visual pigment deactivation does not appear to protect against apoptosis; however, excess flow of opsin into the deactivation pathway may actually increase susceptibility to stress-induced cell death similar to some forms of retinal degeneration.
authors.affiliation: Ross Eye Institute, State University of New York at Buffalo, New York, USA.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Whitcomb
authors.forename: Keisuke
authors.initials: K
authors.lastname: Sakurai
authors.forename: Bruce M
authors.initials: BM
authors.lastname: Brown
authors.forename: Joyce E
authors.initials: JE
authors.lastname: Young
authors.forename: Lowell
authors.initials: L
authors.lastname: Sheflin
authors.forename: Cynthia
authors.initials: C
authors.lastname: Dlugos
authors.forename: Cheryl M
authors.initials: CM
authors.lastname: Craft
authors.forename: Vladimir J
authors.initials: VJ
authors.lastname: Kefalov
authors.forename: Shahrokh C
authors.initials: SC
authors.lastname: Khani
mesh_terms: D000818:Animals; D017209:Apoptosis; D002470:Cell Survival; D022202:Chromosomes, Artificial, Bacterial; D004594:Electrophysiology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005838:Genotype; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D008027:Light; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D011833:Radiation Injuries, Experimental / Q000201:enzymology* / Q000473:pathology; D012160:Retina / Q000528:radiation effects*; D012162:Retinal Degeneration / Q000201:enzymology* / Q000473:pathology; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D012243:Rhodopsin; D051606:G-Protein-Coupled Receptor Kinase 1; C496762:Grk1 protein, mouse