We hypothesize that RPGR isoforms are partitioned in distinct multiprotein complexes in photoreceptors. To evaluate this hypothesis, we have carried out sequential immunodepletion experiments. We initially used antibodies against two of the RPGR partners, CEP290/NPHP6 and SMC1, in order to immunodeplete RPGR that is part of these complexes from the retinal ciliary extract preparation. The remaining supernatant was subjected to immunoprecipitation (IP) with the anti-RPGRORF15 antibody, followed by immunoblotting to test for the presence or absence of remaining RPGRORF15-interacting proteins (). Even after immunodepletion of CEP290 from the retinal ciliary fraction (), RPGR was still associated with IFT88, KIF3A, and γ-tubulin, but not with SMC1 and SMC3 (). This data suggests that RPGR’s complex with CEP290, SMC1, and SMC3 is distinct from that with IFT88, KIF3A, and γ-tubulin. On the other hand, after SMC1 immunodepletion, RPGR antibody could immunoprecipitate only a fraction of CEP290 from retinal ciliary extract. Similar results were obtained with SMC3 (data not shown).
Fig. 13.1 (a) Schematic representation of the strategy to dissect distinct RPGR-containing multiprotein complexes in photoreceptor cilia. IP: Immunoprecipitation; Ab: antibody, (b) Protein extract (~150 µg) was subjected to immunoprecipitation using indicated (more ...)
These observations indicate that RPGR exists in at least three distinct complexes: first with IFT88, KIF3A, and γ-tubulin; second with CEP290, SMC1, and SMC3 and; third with CEP290 and probably other ciliary proteins (). Future detailed analysis of these and additional complexes should assist in dissecting the RPGR function in photoreceptors.
Schematic representation of the putative distinct RPGR complexes that can exist in photoreceptors. Proteins A and B represent as yet unidentified molecular partners that can be part of such complexes