Limited proteolysis differentially modulates the stability and subcellular localization of domains of RPGRIP1 that are distinctly affected by mutations in Leber’s congenital amaurosis

摘要

The retinitis pigmentosa GTPase regulator (RPGR) protein interacts with the retinitis pigmentosa GTPase regulator interacting protein-1 (RPGRIP1). Genetic lesions in the cognate genes lead to distinct and severe human retinal dystrophies. The biological role of these proteins in retinal function and pathogenesis of retinal diseases is elusive. Here, we present the first physiological assay of the role of RPGRIP1 and mutations therein. We found that the monoallelic and homozygous mutations, ΔE1279 and D1114G, in the RPGR-interacting domain (RID) of RPGRIP1, enhance and abolish, respectively, its interaction in vivo with RPGR without affecting the stability of RID. In contrast to RID^WT and RID^D1114G, chemical genetics shows that the interaction of RID^ΔE1279 with RPGR is resistant to various stress treatments such as osmotic, pH and heat-shock stimuli. Hence, RID^D1114G and RID^ΔE1279 constitute loss- and gain-of-function mutations. Moreover, we find that the isoforms, bRPGRIP1 and bRPGRIP1b, undergo limited proteolysis constitutively in vivo in the cytoplasm compartment. This leads to the relocation and accumulation of a small and stable N-terminal domain of ~7 kDa to the nucleus, whereas the cytosolic C-terminal domain of RPGRIP1 is degraded and short-lived. The RID^D1114G and RID^ΔE1279 mutations exhibit strong cis-acting and antagonistic biological effects on the nuclear relocation, subcellular distribution and proteolytic cleavage of RPGRIP1 and/or domains thereof. These data support distinct and spatiotemporal subcellular-specific roles to RPGRIP1. A novel RPGRIP1-mediated nucleocytoplasmic crosstalk and transport pathway regulated by RID, and hence by RPGR, emerges with implications in the molecular pathogenesis of retinopathies, and a model to other diseases.