title: Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2.
abstract: The tip link protein protocadherin 15 (PCDH15) is a central component of the mechanotransduction complex in auditory and vestibular hair cells. PCDH15 is hypothesized to relay external forces to the mechanically gated channel located near its cytoplasmic C terminus. How PCDH15 is coupled to the transduction machinery is not clear. Using a membrane-based two-hybrid screen to identify proteins that bind to PCDH15, we detected an interaction between zebrafish Pcdh15a and an N-terminal fragment of transmembrane channel-like 2a (Tmc2a). Tmc2a is an ortholog of mammalian TMC2, which along with TMC1 has been implicated in mechanotransduction in mammalian hair cells. Using the above-mentioned two-hybrid assay, we found that zebrafish Tmc1 and Tmc2a can interact with the CD1 or CD3 cytoplasmic domain isoforms of Pcdh15a, and this interaction depends on the common region shared between the two Pcdh15 isoforms. Moreover, an interaction between mouse PCDH15-CD3 and TMC1 or TMC2 was observed in both yeast two-hybrid assays and coimmunoprecipitation experiments. To determine whether the Pcdh15-Tmc interaction is relevant to mechanotransduction in vivo, we overexpressed N-terminal fragments of Tmc2a in zebrafish hair cells. Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a-Tmc complex is critical for mechanotransduction. Together, these results identify an evolutionarily conserved association between the fish and mouse orthologs of PCDH15 and TMC1 and TMC2, supporting the notion that TMCs are key components of the transduction complex in hair cells.
authors.affiliation: Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Reo
authors.initials: R
authors.lastname: Maeda
authors.affiliation: Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Katie S
authors.initials: KS
authors.lastname: Kindt
authors.affiliation: Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Weike
authors.initials: W
authors.lastname: Mo
authors.affiliation: Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Clive P
authors.initials: CP
authors.lastname: Morgan
authors.affiliation: Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Timothy
authors.initials: T
authors.lastname: Erickson
authors.affiliation: Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Rachel
authors.initials: R
authors.lastname: Clemens-Grisham
authors.affiliation: Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Barr-Gillespie
authors.affiliation: Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97239 nicolson@ohsu.edu.
authors.forename: Teresa
authors.initials: T
authors.lastname: Nicolson
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000070585:Cadherin Related Proteins; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D006198:Hair Cells, Auditory / Q000502:physiology*; D018069:Hair Cells, Vestibular / Q000502:physiology; D006801:Humans; D040542:Mechanotransduction, Cellular / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010802:Phylogeny; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015027:Zebrafish / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C418976:CDHR15 protein, mouse; C497421:CDHR15 protein, zebrafish; D000070585:Cadherin Related Proteins; D015820:Cadherins; D008565:Membrane Proteins; D011498:Protein Precursors; C514628:TMC1 protein, mouse; C000592940:TMC1 protein, zebrafish; C575755:TMC2 protein, mouse; C000592941:TMC2a protein, zebrafish; D029961:Zebrafish Proteins
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