Usherin, the defective protein in Usher syndrome type IIA, is likely to be a component of interstereocilia ankle links in the inner ear sensory cells

Usher syndrome type IIa (USH2A) combines moderate to severe congenital hearing impairment and retinitispigmentosa. It is the most common genetic form of USH.USH2A encodes usherin, which was previouslydefined as a basement membrane protein. A much larger USH2A transcript predicted to encode a transmem-brane (TM) isoform was recently reported. Here, we address the role of TM usherin in the inner ear. Analysisof the usherin alternative transcripts in the murine inner ear revealed the existence of several predicted TMusherin isoforms with modular ectodomains of different lengths. In addition, we identified in the usherin cyto-plasmic region a predicted 24 amino acid peptide, derived from a newly defined exon that is predominantlyexpressed in the inner ear but not in the retina. In mouse and rat inner ears, we show that TM usherin is pre-sent at the base of the differentiating stereocilia, which make up the mechanosensitive hair bundles receptiveto sound. The usherin immunolabeling is transient in the hair bundles of cochlear hair cells (HCs), but per-sists in mature hair bundles of vestibular HCs. Several lines of evidence support the involvement of TM ush-erin in the composition of the ankle links, a subset of filamentous lateral links connecting stereocilia at thebase. By co-immunoprecipitation and in vitro binding assays, we establish that the usherin cytodomain canbind to whirlin and harmonin, two PDZ domain-containing proteins that are defective in genetic forms of iso-lated deafness and USH type I, respectively. These PDZ proteins are suitable to provide the anchoring ofinterstereocilia lateral links to the F-actin core of stereocilia. Our results strongly suggest that congenitaldeafness in USH type I and type II shares similar pathogenic mechanisms, i.e. the disruption of hairbundle links-mediated adhesion forces that are essential for the proper organization of growing hair bundles.INTRODUCTIONUsher syndrome (USH) is the most frequent cause of heredi-tary deaf–blindness in humans. Three clinical subtypes aredistinguished on the basis of differences in the severity ofhearing impairment and the presence of vestibular dysfunction,whereas progressive visual loss due to retinitis pigmentosawith variable age of onset occurs in all three USH types.USH type II (USH2) is characterized by moderate congenitalhearing loss and normal vestibular function (1). Three differ-ent USH2 loci, USH2A–C, have been defined by linkageanalysis of affected families (2–4). Mutations in the geneencoding usherin underlie USH2A, the most commongenetic form of USH (5,6). Four main types of domainscompose the originally identified USH2A protein, hereafterreferred to as extracellular (EC) usherin: (i) an N-terminaldomain with homology to the laminin globular domainpresent in thrombospondin (TSPN-LG) (7), (ii) a commonN-terminal globular feature of laminins and laminin-relatedproteins called the LN module (8), (iii) a series of 10 rod-like laminin EGF-like (LE) modules (9) and (iv) four fibronec-tin type III (FnIII) repeats (Fig. 1A). Another USH2A