Identification of visual arrestin (S-antigen) in retinal pigmented epithelial cells.
OBJECTIVE
Inactivation of photolyzed rhodopsin requires phosphorylation of this receptor and binding of the 48 kDa regulatory protein arrestin (S-antigen). Arrestin is also to cause an autoimmune disease, uveoretinits, that resembles uveitis in humans. In this study we demonstrate the presence of visual arrestin in retinal pigment epithelial cells (RPE) in culture.


METHODS
Bovine RPE were isolated. Mouse and rat monoclonal and rabbit polyclonal antibodies against visual arrestin, and a synthetic peptide "GFLGELTSSEVATEVPFRLM" (a pathogenic sequence corresponding to residues 340 to 359 of human visual arrestin), and rabbit polyclonal antibody against the specific peptide "EDPDTAKESFQ" for bovine visual arrestin were used to detect arrestin in RPE cells. Using visual arrestin specific primer, RT-PCR of RNA from RPE was performed.


RESULTS
By western blots analysis a 48 kDa protein, corresponding to visual arrestin was detected with both mAb and polyclonal antibodies in extracts of RPE cells. RT-PCR analysis of RNA from RPE cells confirmed the presence of arrestin mRNA of predicted 377 bp and exhibited 100% homology with visual arrestin 48 kDa.


CONCLUSIONS
Visual arrestin proteins present in RPE may be involved in the desensitization of G-protein-coupled receptors in RPE cells and in arrestin uveopathogenesis.
Nicolas
C
C
Faculté de Médecine Paris VI, INSERM U 86/E9912, Paris, France.
Ghedira
I
I
Stiemer
R
R
Teufel
B
B
Hecquet
C
C
Faure
J P
JP
Mirshahi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D019393:Arrestin / Q000032:analysis*; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010857:Pigment Epithelium of Eye / Q000737:chemistry* / Q000166:cytology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011917:Rats, Inbred Lew; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D019393:Arrestin; D017931:DNA Primers; D010446:Peptide Fragments; D012333:RNA, Messenger