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Atsuhiro Kanda, Kousuke Noda, Susumu Ishida; Identification of (pro)renin receptor/Atp6ap2-binding proteins in the retina of adult mice. Invest. Ophthalmol. Vis. Sci. 2014;55(13):354.
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(Pro)renin receptor [(P)RR] (also called Atp6ap2), identified as a molecule existing in the major organs but not in the circulation, has attracted growing attention due to its diverse roles in tissue renin-angiotensin system (RAS) and in fundamental cellular physiology related to vacuolar H+-ATPase (v-ATPase). The purpose of this study is to determine retinal proteins bound to Atp6ap2/(P)RR in adult mice.
We performed immunoprecipitation and mass spectrometry (MS) experiments to identify candidate interacting proteins of Atp6ap2/(P)RR. Pre-cleared C57BL/6J retina lysates with protein G beads were immunoprecipitated with normal IgG or anti-Atp6ap2 antibodies. The beads were washed, and protein samples were then eluted by boiling in sample buffer and separated by SDS-PAGE. Eluted samples observed by silver staining that were identified as being unique to anti-Atp6ap2 antibody as compared to normal IgG have been examined by MS.
MS analysis results showed candidate proteins for possible Atp6ap2/(P)RR binding partners include Ablim2, Atp6v0d1 and Rpl6. We confirmed those protein interaction with co-transfection/co-immunoprecipitation experiments and co-localizations in mouse retina with immunofluorescence.
Our current findings may help elucidate various physiological activities of Atp6ap2/(P)RR in the retina of adult mice. Given that Atp6ap2/(P)RR functions as a cell polarity determinant required for retinal lamination during embryonic development in mice (Kanda A et al. J Neurosci. 2013), Atp6ap2/(P)RR is thought to be involved in multiple physiological and pathological events in the eye, besides the v-ATPase function and tissue RAS activation.
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