Molecular Analysis of Human Mutations on the Interactions Between RPGR, RPGRIP1, and PDE

Abstract

Abstract: : Purpose: A large number of allelic–specific mutations in Retinitis Pigmentosa GTPase Regulator (RPGR) lead to a number of distinct retinal dystrophies and a few of these co–segregate with systemic disorders of apparently variable genetic penetrance. Two major RPGR isoforms were reported and herein designated, RPGR_1–19 and RPGR_ORF15. Human mutations affect just the latter or both isoforms. The conserved RCC1–homologous domain (RHD) of RPGR was reported to interact with the Retinitis Pigmentosa GTPase Regulator–Interacting Protein 1 (RPGRIP1) and the Δ subunit of phosphodiesterase (ΔPDE) and numerous misensense mutations were found in RHD of RPGR. Homozygous, heterozygous and monoallelic mutations in RPGRIP1 were found to lead to Leber congenital amaurosis (LCA) but no mutations have been reported in ΔPDE. Two mutations, D1114G and ΔE1279, have been found in the RPGR–interacting domain (RID) of RPGRIP1. The former has been reported in homozygous and heterozygous states, while the latter only in a monoallelic form. This study aims at determining the molecular basis of the mutation effects in the interactions between RPGR, RPGRIP1, and ΔPDE. Methods: We employed genetic interaction–trap, chemical genetic and biochemical assays to evaluate the mutation effects in RPGR, RPGRIP1, and ΔPDE and interactions between these. Results:The LCA mutations, D1114G and ΔE1279, in the RID of RPGRIP1 were found, respectively, to abolish completely and enhance significantly (by ∼ 40%) the strength of the interaction with the RHD of RPGR and without affecting the stability of the the RID of RPGRIP1. The latter mutation also confers a significant resilient phenotype to the interaction between RPGR and RPGRIP1 upon exposure of these to selective and chemical pressures in vivo. In contrast to a previous report, we found that the interaction between RPGR_1–19 and RPGR_ORF15, and ΔPDE, was significantly stronger with RPGR_1–19 and its C–terminal half domain, but not its RHD domain. Yet, many but not all missense mutations in RHD of RPGR_1–19 abolished its interaction with ΔPDE. Finally, ∼ 34 mutations were introduced mostly in the N–terminal half of RPGR and these were found to affect differentially the interaction between RPGR and, ΔPDE and RPGRIP1. Conclusions: The data support the existence of distinct roles between RPGR_1–19 and RPGR_ORF15, and their molecular partners. Moreover, mutations in RPGR and RPGRIP1 generate distinct molecular phenotypes, which may reflect a diversity of pathomechanisms leading to retinal dystrophies associated with XlRP3 and LCA.

Keywords: genetics • retinal degenerations: hereditary • proteins encoded by disease genes