Molecular Analysis of the Supramolecular Usher 1 Protein Complex in the Retina

Abstract

Abstract: : Purpose: Human Usher syndrome (USH) is the most common form of deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and prepubertal-onset retinitis pigmentosa. The aim of our project is to analyze the molecular and cellular function of the identified USH1 proteins in the vertebrate retina. Methods: In vitro binding assays (e.g. GST-pull downs) are applied. Isoform-specific antibodies to USH1 proteins are used in Western blot analysis of subcellular photoreceptor fractions and immunofluorescence microscopy of the retina. Results: In vitro F-actin binding assays and transfection studies reveal that harmonin (USH1C) is a potent actin bundling protein. GST-pull downs and co-transfections reveal that harmonin acts as a scaffold protein for USH1 proteins binding the molecular motor myosin VIIa (USH1B) via its PDZ-1 and the cell-cell adhesion molecule cadherin 23 (USH1D) via its PDZ-2. All three proteins are expressed in the neuronal retina. Immunocytochemistry and biochemical analyses of fractionated photoreceptor compartments reveal that the identified components of this supramolecular USH1-complex co-localize at the ribbon synapse of retinal photoreceptor cells. Conclusions: At the photoreceptor synapse, the USH1-complex may contribute to the cortical cytoskeletal matrices of the pre- and postsynaptic regions which are thought to play a fundamental role in the structural and functional organization of the synaptic junction. The scaffold protein harmonin may bridge the motor activity of myosin VIIa involved in endo- or exocytotic processes at the synapse with the cell-cell adhesion complex generated by cadherin 23 and other transmembrane proteins (e.g. the myosin VIIa ligand vezatin). Dysfunction of any of the USH1-complex partners may result in synaptic dysfunction causing retinitis pigmentosa, the clinical phenotype in the retina of USH1 patients.

Keywords: photoreceptors • synapse • proteins encoded by disease genes