title: Fates of Cdh23/CDH23 with mutations affecting the cytoplasmic region.
abstract: BUS/Idr mice carrying a mutant waltzer allele (vbus) 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 vbus/vbus tissues, while the antibodies immunoreacted with approximately 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 vbus/vbus 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.
authors.affiliation: Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan. yonez@inst-hsc.jp
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yonezawa
authors.forename: Norio
authors.initials: N
authors.lastname: Yoshizaki
authors.forename: Takashi
authors.initials: T
authors.lastname: Kageyama
authors.forename: Takayuki
authors.initials: T
authors.lastname: Takahashi
authors.forename: Mamoru
authors.initials: M
authors.lastname: Sano
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Tokita
authors.forename: Shigeo
authors.initials: S
authors.lastname: Masaki
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Inaguma
authors.forename: Atsuko
authors.initials: A
authors.lastname: Hanai
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Sakurai
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yoshiki
authors.forename: Moriaki
authors.initials: M
authors.lastname: Kusakabe
authors.forename: Akihiko
authors.initials: A
authors.lastname: Moriyama
authors.forename: Atsuo
authors.initials: A
authors.lastname: Nakayama
mesh_terms: D000818:Animals; D000070585:Cadherin Related Proteins; D015820:Cadherins / Q000737:chemistry / Q000235:genetics*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry*; D015870:Gene Expression; D006579:Heterozygote; D006720:Homozygote; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D009154:Mutation / Q000235:genetics*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: C442480:CDH23 protein, human; D000070585:Cadherin Related Proteins; D015820:Cadherins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins