Identification and subcellular localization of the RP1 protein in human and mouse photoreceptors.
OBJECTIVE
Mutations in the RP1 gene account for 6% to 10% of autosomal dominant retinitis pigmentosa (adRP). Previous studies have shown that the RP1 gene is expressed specifically in photoreceptor cells. So far, little is known about the RP1 protein or how mutations in RP1 lead to photoreceptor cell death. The goal of this study was to identify the RP1 protein and investigate its location in photoreceptor cells.


METHODS
A combination of RT-PCR and rapid amplification of cDNA ends (RACE) was used to isolate the full-length mouse Rp1 cDNA. Antibodies against different regions of the predicted mouse Rp1 protein were generated. Western blot analyses were used to identify the RP1/Rp1 proteins. The subcellular location of RP1 in human and mouse retinas was determined by immunostaining retinal sections.


RESULTS
The full-length mouse Rp1 cDNA is 6944 bp, encoding a predicted protein of 2095 amino acids. Rp1 was found to be a soluble protein of approximately 240 kDa, consistent with predictions based on the cDNA sequence. Immunofluorescence analyses revealed that both the human RP1 and mouse Rp1 proteins are specifically localized in the connecting cilia of rod and cone photoreceptors.


CONCLUSIONS
The presence of RP1/Rp1 in connecting cilia suggests that it may participate in transport of proteins between the inner and outer segments of photoreceptors or in maintenance of cilial structure. This study forms the basis for further investigation of the function of RP1 in retina and the mechanism by which mutations in RP1 lead to photoreceptor cell death.
Liu
Qin
Q
F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Zhou
Jie
J
Daiger
Stephen P
SP
Farber
Debora B
DB
Heckenlively
John R
JR
Smith
Julie E
JE
Sullivan
Lori S
LS
Zuo
Jian
J
Milam
Ann H
AH
Pierce
Eric A
EA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005784:Gene Amplification; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins; D012174:Retinitis Pigmentosa / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D005136:Eye Proteins; D008869:Microtubule-Associated Proteins; C120042:RP1 protein, human; D011993:Recombinant Fusion Proteins