Molecular Linkage Between Usher Syndrome 1 and 2 by Interacting Within Supramolecular Usher Protein Complexes

Abstract

Abstract: : Purpose: The human Usher syndrome (USH) is the most common form of deaf–blindness characterized by congenital deafness and retinitis pigmentosa. Based on the degree of clinical symptoms, USH is distinguished into three types USH1, USH2 and USH3 which show genetic heterogeneity with at least 11 chromosomal loci. All USH1–proteins identified have been shown to interact via the USH1C protein harmonin, a scaffold protein containing up to three PDZ–domains which are responsible for protein–protein binding. The aim of our present study was to validate whether or not USH2–gene products are also part of this USH–protein complexes in the mammalian retina. Methods: Antibodies against bacterial expressed USH proteins were generated. Affinity purified antibodies were used in Western blot and immunocytochemical analyses of rodent and human retinas. In vitro binding assays (co–immunoprecipitation, GST–pull downs, yeast 2–hybrid assays) were applied. Results: In sequence analyses, class I PDZ–interacting motifs were identified in cytoplasmic C–terminal of all three USH2–proteins, namely the transmembrane protein usherin (USH2A), the ion exchanger NBC3 (USH2B), and the very large 7–transmembrane G–protein coupled receptor VLGR1b (USH2C). In all performed in vitro binding assays, cytoplasmic domains of USH2–proteins interacted with full length harmonin a1. Further assays revealed that these interactions between harmonin and the 3 USH2–proteins occur via harmonin's PDZ–1 motif. Western blot analysis of retinal extracts demonstrated that all binding partners were expressed in the neuronal retina. Cytochemical subcellular localization of USH2– and USH1–proteins revealed co–localization of all USH–proteins analyzed at the ribbon synapse of photoreceptor cells. Conclusions: The results of our present study provide the first evidence for a common pathophysiological pathway of USH1 and USH2. Proteins of both USH–types can act as components of supremolecular USH–complexes molecularly linked via the scaffold protein harmonin. In photoreceptor cells, the USH–complex may contribute to the cortical cytoskeletal matrices of pre– and postsynaptic regions which are thought to play a fundamental role in the structural and functional organization of the synaptic junction. Dysfunction of any of the USH–complex partners may result in synaptic dysfunction causing retinitis pigmentosa, the common retinal phenotype in USH1 and 2–patients.

Keywords: proteins encoded by disease genes • photoreceptors • synapse