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J. Biol. Chem., Vol. 280, Issue 39, 33580-33587, September 30, 2005

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RPGR-ORF15, Which Is Mutated in Retinitis Pigmentosa, Associates with SMC1, SMC3, and Microtubule Transport Proteins^*

Hemant Khanna, Toby W. Hurd1, Concepcion Lillo¶, Xinhua Shu||, Sunil K. Parapuram, Shirley He, Masayuki Akimoto**, Alan F. Wright||, Ben Margolis2, David S. Williams¶, and Anand Swaroop3

From the Departments of Ophthalmology & Visual Sciences and Human Genetics, University of Michigan, Ann Arbor, Michigan 48105, the Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109, the ^¶ Departments of Pharmacology and Neurosciences, School of Medicine, University of California, La Jolla, California 92093, the ^||MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, United Kingdom, and the ^**Translational Research Center, Kyoto University Hospital, Sakyo-ku, Kyoto, 606-8507 Japan

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of photoreceptor connecting cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150^Glued, and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia.

Received for publication, May 27, 2005 , and in revised form, July 19, 2005.

^* This work was supported in part by National Institutes of Health Grants EY07961, EY07003, EY13408, EY12598, and DK069605 and the Foundation Fighting Blindness and Research to Prevent Blindness. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Polycystic Kidney Disease Foundation Grant 92a2f.

² Investigator of the Howard Hughes Medical Institute.

³ Harold F. Falls Collegiate Professor and Research to Prevent Blindness Senior Scientific Investigator. To whom correspondence should be addressed: Dept. of Ophthalmology and Visual Sciences, University of Michigan, W.K. Kellogg Eye Center, 1000 Wall St., Ann Arbor, MI 48105. Tel.: 734-763-3731; Fax: 734-647-0228; E. mail: swaroop{at}umich.edu.

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