Magnification, 60. Double immunolabeling was also used to verify the location of RP1 in the rods of human retinas. acids. Rp1 was found to be a soluble protein of approximately 240 kDa, consistent with predictions based on the cDNA sequence. GS-626510 Immunofluores-cence 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 GS-626510 structure. This study forms the basis for further investigation of the function of RP1 in retina and the mechanism by which mutations in em RP1 /em lead to photoreceptor cell death.( em Invest Ophthalmol GS-626510 Vis Sci /em . 2002;43:22C2032) Retinitis pigmentosa (RP) is a group of inherited retinal degeneration disorders characterized by night blindness, progressive loss of peripheral vision, and characteristic pigmentary retinopathy. RP is the most common inherited form of blindness, affecting more than 100,000 people in the United States and 1.5 million people worldwide.1 In addition to variations in clinical phenotype, RP is genetically heterogeneous and can be inherited by autosomal dominant (ad), autosomal recessive (ar), or X-linked transmission as well as a rare digenic mode.1,2 adRP accounts for approximately 15% to 20% of RP cases. Linkage analyses have demonstrated 11 genetic loci for adRP to date.2,3 So far, the genes at four of these loci have been identified.3 The em RP1 /em gene was the fourth dominant RP gene to be identified,4C6 after em RHO /em , em RDS /em , and em NRL /em , which encode rhodopsin, peripherin/RDS, and NRL, respectively.7C9 The em RP1 /em gene is located on chromosome 8q12 and consists of four exons with an open reading frame of 6468 bp, encoding a predicted protein of 2156 amino acids, mostly by exon 4 (788C6468 bp). The em RP1/Rp1 /em gene is usually expressed only in the photoreceptor cells of the retina, as determined by Northern blot analysis4C6 and in situ hybridization.4 Analysis of homology between human RP1 and other known proteins demonstrates that this N-terminal portion of RP1 is related to dou-blecortin (DCX), which is believed to be involved in directing neuronal migration during development of the central nervous system.10 So far, 20 disease-causing mutations have been identified in the em RP1 /em gene.4C6,11C13 These are either nonsense or frame-shift mutations that cluster within a region extending from codons 658-1053 in exon 4. All these mutant alleles would encode truncated proteins without the carboxy 50% to 70% of RP1. Together these mutations account for approximately 6% to 10% of adRP cases in different ethnically diverse populations.4,6,11C13 The most common mutation in em RP1 /em , Arg677Ter, is present in approximately 3% of patients with adRP in the United States,4 constituting the third most common adRP mutation, after the Pro23His (9% of cases) and Pro347Leu (4% of cases) mutations in the rhodopsin gene.14 These findings indicate that this RP1 protein plays an important, although as yet unknown, role in photoreceptor function. To elucidate the function of the RP1 protein and to gain insight into the mechanisms by which mutations in em RP1 /em cause retinal degeneration, we cloned and sequenced the full-length mouse em Rp1 /em cDNA. Based on the amino acid sequence predicted from em Rp1 /em cDNA, we generated antibodies against mouse Rp1 fusion proteins. These antibodies were used to detect the RP1/Rp1 proteins by immunoblotting and to localize the RP1/ Rp1 proteins in human and mouse retinas by immunostaining. Our results show that this RP1/Rp1 protein is located in the connecting cilia of rod and cone photoreceptor Foxd1 cells, making it the second protein specifically localized in this important structure of photoreceptors. METHODS Animals and Human Tissues This research adhered to the tenets of the Declaration of Helsinki, the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research, and the guidelines of the University of Pennsylvania in Animal Care and Use. C57Bl/6J mice and Sprague-Dawley rats were obtained from Jackson Laboratories (Bar Harbor, ME). Frozen cow retinas were purchased from JA Lawson, Inc. (Lincoln, NE). Normal human frozen eyes were provided by the Foundation Fighting Blindness Eye Lender at the Scheie Vision Institute (Philadelphia, PA). Isolation.
