Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis

Abstract

Purpose: The human Usher syndrome (USH) is the most common cause of combined deaf-blindness. The diverse USH proteins are integrated in protein networks by scaffolds including the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain). Previous data indicated that SANS participates in microtubule-based transport modules. Here we searched for new SANS interacting partners to get further insights into the role of SANS protein networks and their regulation in photoreceptor cells.

Methods: Y2H screens of retinal cDNA libraries, validation of the putative interaction partners by independent complementary assays: GST-pull downs, membrane targeting assays, co-immunoprecipitations and PLA. Immunofluorescence and immunoelectron microscopy for subcellular localization of network proteins; endocytosis and phosphorylation assays combined with knock down experiments.

Results: In Y2H screens we identified the MAGUK protein MAGI2 as a putative interactor of SANS. We affirmed this interaction and pinpointed the binding of MAGI2-PDZ5 to a novel internal PBM-motif in the SAM domain of SANS. We demonstrated that the assembly is regulated by the phosphorylation of this motif in SANS by the protein kinase CK2. We showed that Magi2-mediated, clathrin-dependent endocytosis is negatively regulated by binding of phosphorylated SANS to Magi2. Further, knock-down experiments revealed that SANS and Magi2-mediated endocytosis regulated aspects of ciliogenesis. Finally, we demonstrated the localization of the complex in the periciliary membrane complex facing the ciliary pocket of retinal photoreceptor cells.

Conclusions: Our data indicate that endocytosis processes may not only contribute to photoreceptor cell homeostasis, but also counterbalance the periciliary membrane delivery accompanying the exocytosis processes for the cargo vesicle delivery. In USH1G patients, mutations in SANS eliminate Magi2 binding site and thereby deregulate endocytosis, subsequently leading to defects in ciliary transport modules, and ultimately disrupt photoreceptor cell function inducing retinal degeneration.

Keywords: 648 photoreceptors • 659 protein structure/function • 695 retinal degenerations: cell biology