Fates of Cdh23/CDH23 with mutations affecting the cytoplasmic region

Abstract BUS/Idr mice carrying a mutant waltzer allele ( v bus ) are characterized by splayed hair bundles in inner ear sensory cells, providing a mouse homolog of USH1D/DFNB12. RT‐PCR‐based screening for the presence of mutations in mouse Cdh23 , the gene responsible for the waltzer phenotype, has identified a G>A mutation in the donor splice site of intron 67 (Cdh23:c.9633+1G>A: GenBank AF308939.1), indicating that two altered Cdh23 molecules having intron‐derived COOH‐terminal structures could be generated in BUS mouse tissues. Immunochemical analyses with anti‐Cdh23 antibodies showed, however, no clear Cdh23‐related proteins in v bus / v bus tissues, while the antibodies immunoreacted with ∼350 kDa proteins in control mice. Immunofluorescent experiments revealed considerable weakening of Cdh23 signals in sensory hair cell stereocilia and Reissner's membrane in the v bus / v bus inner ear, and transmission electron microscopy demonstrated abundant autophagosome/autolysosome vesicles, suggesting aberrant Cdh23:c.9633+1G>A‐derived protein‐induced acceleration of lysosomal bulk degradation of proteins. In transfection experiments, signal sequence‐preceded FLAG‐tagged transmembrane plus cytoplasmic regions (TMCy) of tissue‐specific Cdh23(±68) isoforms were localized to filamentous actin‐rich protrusions and the plasma membrane of cultured cells, whereas FLAG‐TMCy:c.9633+1G>A proteins were highly insoluble and retained in the cytoplasm. In contrast, FLAG‐tagged TMCy:p.Arg3175His and human TMCy:c.9625_9626insC forms were both localized to the plasma membrane in cultured cells, allowing prediction that USH1D‐associated CDH23:p.Arg3175His and CDH23:c.9625_9626insC proteins could be transported to the plasma membrane in vivo. The present results thus suggest different fates of CDH23/Cdh23 with mutations affecting the cytoplasmic region. Hum Mutat 27(1), 88–97, 2005. © 2005 Wiley‐Liss, Inc.