Deposition of exon-skipping splice isoform of human retinal G protein-coupled receptor from retinal pigment epithelium into Bruch's membrane.
OBJECTIVE
Human retina and retinal pigment epithelium (RPE) express a relatively abundant mRNA that encodes an extraneous splice isoform of the RPE retinal G protein-coupled receptor (RGR) opsin. In this study, we investigate this exon-skipping RGR splice isoform (RGR-d) in separated neural retina and RPE cells of human donors of various ages.


METHODS
We used mass spectrometry, sensitive western blot assay, immunohistochemical localization and real-time RT-PCR to analyze RGR-d.


RESULTS
Western blot assay detected the RGR-d protein in the neural retina of all donors analyzed. Mass spectrometric analysis of the immunoreactive proteins independently confirmed the presence of RGR-d. In contrast, RGR-d protein in the RPE of most donors was barely detectable by western blot assay, even though expression of RGR-d mRNA was confirmed by amplification of RGR-d transcripts in both the RPE and neural retina. Quantitative real-time RT-PCR assays showed that RGR-d/RGR mRNA transcript ratios were about 0.17 and about 0.33 in the RPE and neural retina, respectively. Immunohistochemical localization studies revealed that the RGR-d epitope was present near the basal boundary of RPE cells and primarily in the extracellular areas of Bruch's membrane, adjacent choriocapillaris, and intercapillary region of both young and older donors. Positive immunostaining was seen in the drusen of older individuals.


CONCLUSIONS
The RGR-d protein is a common mutant form of human RGR that can be identified in donor eyes by mass spectrometry. These results indicate that after RGR-d is synthesized, the RGR-d epitope is released at the basal surface of the RPE and deposited into Bruch's membrane in human eyes throughout adult life.
Doheny Eye Institute, Los Angeles, CA 90033, USA.
Meng-Yin
MY
Lin
Harold
H
Kochounian
Roger E
RE
Moore
Terry D
TD
Lee
Narsing
N
Rao
Henry K W
HK
Fong
D000293:Adolescent; D000328:Adult; D000368:Aged; D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D016570:Bruch Membrane / Q000166:cytology / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D005091:Exons / Q000235:genetics*; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D000939:Epitopes; D005136:Eye Proteins; C089754:G protein-coupled receptor RGR; D020033:Protein Isoforms; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled