Structural and functional plasticity of subcellular tethering, targeting and processing of RPGRIP1 by RPGR isoforms

Abstract

SummaryMutations affecting the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) interactome cause syndromic retinal dystrophies. RPGRIP1 interacts with the retinitis pigmentosa GTPase regulator (RPGR) through a domain homologous to RCC1 (RHD), a nucleotide exchange factor of Ran GTPase. However, functional relationships between RPGR and RPGRIP1 and their subcellular roles are lacking. We show by molecular modeling and analyses of RPGR disease-mutations that the RPGR-interacting domain (RID) of RPGRIP1 embraces multivalently the shared RHD of RPGR1–19 and RPGRORF15 isoforms and the mutations are non-overlapping with the interface found between RCC1 and Ran GTPase. RPGR disease-mutations grouped into six classes based on their structural locations and differential impairment with RPGRIP1 interaction. RPGRIP1α1 expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1α1 self-aggregation ensue. RPGR1–19 localizes to the endoplasmic reticulum, whereas RPGRORF15 presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1α1. Disease mutations in RPGR1–19, RPGRORF15, or RID of RPGRIP1α1, singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1α1 with RPGR1–19 or RPGRORF15 in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGRORF15, but not RPGR1–19, protects the RID of RPGRIP1α1 from limited proteolysis. These studies define RPGR- and cell-type-dependent targeting pathways with structural and functional plasticity modulating the expression of mutations in RPGR and RPGRIP1. Further, RPGR isoforms distinctively determine the subcellular targeting of RPGRIP1α1, with deficits in RPGRORF15-dependent intracellular localization of RPGRIP1α1 contributing to pathomechanisms shared by etiologically distinct syndromic retinal dystrophies.

RPGR: Its role in photoreceptor physiology, human disease, and future therapies

“…The RPGR Ex1-19 transcript encodes a predicted 90 kDa protein with 19 exons of the gene transcribed. Exons 1 to 10 of RPGR Ex1-19 encode an RCC1-like domain ( Meindl et al., 1996 ; Wätzlich et al., 2013 ) and the C-terminus has an isoprenylation motif (CAAX), suggesting that this isoform is membrane bound, consistent with its reported attachment to endoplasmic reticulum membranes in addition to its presence at CC ( Patil et al., 2012a ). Yan et al.…”

“…The protein products of the two major human RPGR alternative transcripts have been extensively studied ( Fig. 1 a) ( Meindl et al., 1996 ; Roepman et al., 1996 ; Vervoort et al., 2000; Mavlyutov et al., 2002; Hong et al., 2003; Patil et al., 2012a ).…”

“… Yan et al. (1998) suggested a Golgi localisation for RPGR but this has not been confirmed ( Patil et al., 2012a ). No disease-causing mutations have been reported in exons 16 to 19.…”

Differential Loss of Prolyl Isomerase or Chaperone Activity of Ran-binding Protein 2 (Ranbp2) Unveils Distinct Physiological Roles of Its Cyclophilin Domain in Proteostasis

“…(equivalent residues of the mouse sequence), fused in-frame to GST or mRFP were produced from mutant amplicons of bovine cDNA constructs (58) by a two-step PCR strategy with one pair of complementary primers comprising the point mutation(s) and another pair flanking the mutated primers upstream and downstream as described elsewhere (71). GST-fused and thrombin-cleaved (GST-free) recombinant proteins were expressed, purified, and analyzed exactly as described elsewhere (72).…”

High Symmetry of Visual Acuity and Visual Fields in RPGR-Linked Retinitis Pigmentosa

“…Aside from the constitutive variant RPGR , which is widely expressed throughout the body, 7-9 over 10 splice variants of RPGR exist. [10][11][12][13] The RPGR ORF15 variant is abundantly expressed in the retina 8,[14][15][16][17] where it is localized at the connecting cilium of the photoreceptors 11,18 and interacts with several proteins through a regulator of chromosome condensation (RCC1)-like domain at its N-terminus, most notably the RPGR-interacting protein (RPGRIP1). 19 RPGR ORF15 shares exons 1-14 and part of exon 15 with RPGR , which includes the abovementioned RCC1-like domain at the N-terminus, but ORF15, the terminal exon, is unique to RPGR ORF15 .…”