RPGR protein complex regulates proteasome activity and mediates store-operated calcium entry.

Sarita Rani Patnaik,Susan Chalmers,Helen May-Simera,Deva Krupakar Kusuluri,James Reilly,Xun Zhang,Lincoln Biswas,Xinzhi Zhou,Saeed Akhtar,John G Mccarron,Xinhua Shu

doi:10.18632/oncotarget.25259

Abstract

Ciliopathies are a group of genetically heterogeneous disorders, characterized by defects in cilia genesis or maintenance. Mutations in the RPGR gene and its interacting partners, RPGRIP1 and RPGRIP1L, cause ciliopathies, but the function of their proteins remains unclear. Here we show that knockdown (KD) of RPGR, RPGRIP1 or RPGRIP1L in hTERT-RPE1 cells results in abnormal actin cytoskeleton organization. The actin cytoskeleton rearrangement is regulated by the small GTPase RhoA via the planar cell polarity (PCP) pathway. RhoA activity was upregulated in the absence of RPGR, RPGRIP1 or RPGRIP1L proteins. In RPGR, RPGRIP1 or RPGRIP1L KD cells, we observed increased levels of DVl2 and DVl3 proteins, the core components of the PCP pathway, due to impaired proteasomal activity. RPGR, RPGRIP1 or RPGRIP1L KD cells treated with thapsigargin (TG), an inhibitor of sarcoendoplasmic reticulum Ca2+- ATPases, showed impaired store-operated Ca2+ entry (SOCE), which is mediated by STIM1 and Orai1 proteins. STIM1 was not localized to the ER-PM junction upon ER store depletion in RPGR, RPGRIP1 or RPGRIP1L KD cells. Our results demonstrate that the RPGR protein complex is required for regulating proteasomal activity and for modulating SOCE, which may contribute to the ciliopathy phenotype.

Highlights

Primary cilia are microtubule-based, non-motile, solitary organelles emerging from the surface of many vertebrate cells
Quantitative real-time PCR and Western blotting were performed at 48 h post transfection to verify the efficiency of retinitis pigmentosa GTPase regulator (RPGR), RPGR-interacting protein-1 (RPGRIP1) or RPGRIP1L depletion and confirmed that the three genes were effectively knocked down (Supplementary Figure 1)
We measured the length of the actin bundles in the photoreceptors in these mice and found that they were longer in RPGR knockout mice than in wildtype control mice (Figure 2A, 2B)

Summary

Introduction

Primary cilia are microtubule-based, non-motile, solitary organelles emerging from the surface of many vertebrate cells. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the major cause of XLRP [3,4,5]. Deletion of RPGR results in a slower retinal degeneration [16, 17], while loss of RPGRIP1 leads to an early onset retinal degeneration with abnormal development of outer segments [18, 19]. Global deletion of RPGRIP1L in mouse causes mid-gestation lethality with cilia defects in multiple organs, corresponding closely to the clinical phenotype observed in MKS [14]. Together these data suggest that RPGR, RPGRIP1 and RPGRIP1L are critical in ciliary homeostasis

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Results

Conclusion