Abstract
Purpose: :
Mutations affecting components of the Retinitis Pigmentosa GTPase Regulator Protein 1 (RPGRIP1) interactome are implicated in syndromic retinal dystrophies by elusive mechanisms. The Retinitis Pigmentosa GTPase Regulator (RPGR) and RPGRIP1 play also central roles in the maintenance or genesis of the outer segments of photoreceptors. RPGR interacts with RPGRIP1 via its RCC1-homologous domain (RHD). The aim of this work is to determine structural, functional and pathobiological relationships between RPGR and RPGRIP1α1 isoforms with and without disease mutations.
Methods: :
We used a variety of interdisciplinary approaches, such as molecular modeling, structure-function analyses, mutagenesis, cell-based assays combined with time-lapse microscopy and biochemistry, to examine the interplay between various wild-type and mutant RPGR and RPGRIP1α1 isoforms.
Results: :
Molecular modeling of RHD of RPGR to RCC1 shows that disease mutations in RHD outline a contact interface distinct from that found between RCC1 and RAN GTPase. Quantitative yeast two-hybrid assays show that disease-causing mutations in RHD or ORF15 domains differentially impair RPGR interaction with RPGRIP1. Cell and time-lapse microscopy assays support that expression of RPGRIP1α1 isoform alone promotes its profuse self-aggregation, whereas RPGRIP1α1 co-expression with the RPGR1-19 isoform targets RPGRIP1α1 to the Golgi. Conversely, RPGRORF15 co-expression with RPGRIP1α1 promotes its pan-intracellular dispersion and clears-out pre-existing RPGRIP1α1 deposits. Co-expression, but not single expression, of disease mutations in RHD of RPGR isoforms and RID of RPGRIP1α1 abrogates their colocalization and physical association in COS7 cells, whereas mutations singly in any RPGR isoform suffice to promote delocalization from RPGRIP1α1 in a photoreceptor line. RPGRORF15, but not RPGR1-19, protects RPGRIP1α1 from limited C-terminal proteolysis.
Conclusions: :
Rpgr1-19 and RpgrORF15 exert distinct effects on RPGRIP1α1 sorting/processing and they are necessary, but not sufficient in a cell-context-dependent manner, for the coupling to, and subcellular targeting of, RPGRIP1α1. A model emerges by which the structural plasticity and dynamic composition of the RPGRIP1 interactome underlie variations in disease expression. Moreover, RpgrORF15-dependent deficits in post-Golgi trafficking events likely underlie the pathogenesis of XlRP3 and LCA.
Keywords: proteins encoded by disease genes • protein structure/function • mutations