Targeting of the hair cell proteins cadherin 23, harmonin, myosin XVa, espin, and prestin in an epithelial cell model

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Abstract

We have developed an advantageous epithelial cell transfection model for examining the targeting, interactions, and mutations of hair cell proteins. When expressed in LLC-PK1-CL4 epithelial cells (CL4 cells), the outer hair cell protein prestin showed faithful domain-specific targeting to the basolateral plasma membrane. We examined the consequences of mutations affecting prestin activity and assigned a targeting role to the cytoplasmic tail. The stereociliary link protein cadherin 23 (Cdh23) was targeted to the plasma membrane of CL4 cell microvilli, the topological equivalent of stereocilia. In cells coexpressing the Cdh23 cytoplasmic binding protein harmonin, a large fraction of harmonin became colocalized with Cdh23 in microvilli. Using this assay and in vitro protein binding assays, we formulated an alternative model for Cdh23-harmonin binding, in which the primary interaction is between the harmonin N-domain and a 35-residue internal peptide in the Cdh23 cytoplasmic tail. Contrary to a previous model, we found no role for the Cdh23 C-terminal PDZ (PSD-95/Dlg/ZO-1)-binding motif and observed that Cdh23 bound similar levels of harmonin with or without the exon 68 peptide. We also examined two proteins involved in stereocilium elongation. The stereociliary actin-bundling protein espin was targeted to CL4 cell microvilli and caused microvillar elongation, whereas espin with the c.2469delGTCA or c.1988delAGAG human deafness mutation showed defects in microvillar targeting and elongation. The unconventional myosin motor myosin XVa accumulated at the tips of espin-elongated microvilli, by analogy to its location in stereocilia, whereas myosin XVa with the c.4351G>A or c.4669A>G human deafness mutation did not, revealing functional deficits in motor activity.

Original languageEnglish (US)
Pages (from-to)7187-7201
Number of pages15
JournalJournal of Neuroscience
Volume30
Issue number21
DOIs
StatePublished - May 26 2010

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Cadherins
Myosins
Epithelial Cells
Stereocilia
Microvilli
Proteins
Mutation
Deafness
Outer Auditory Hair Cells
Tail
Cell Membrane
Peptides
Protein Binding
Transfection
Actins
Exons
Carrier Proteins
Motor Activity

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

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title = "Targeting of the hair cell proteins cadherin 23, harmonin, myosin XVa, espin, and prestin in an epithelial cell model",
abstract = "We have developed an advantageous epithelial cell transfection model for examining the targeting, interactions, and mutations of hair cell proteins. When expressed in LLC-PK1-CL4 epithelial cells (CL4 cells), the outer hair cell protein prestin showed faithful domain-specific targeting to the basolateral plasma membrane. We examined the consequences of mutations affecting prestin activity and assigned a targeting role to the cytoplasmic tail. The stereociliary link protein cadherin 23 (Cdh23) was targeted to the plasma membrane of CL4 cell microvilli, the topological equivalent of stereocilia. In cells coexpressing the Cdh23 cytoplasmic binding protein harmonin, a large fraction of harmonin became colocalized with Cdh23 in microvilli. Using this assay and in vitro protein binding assays, we formulated an alternative model for Cdh23-harmonin binding, in which the primary interaction is between the harmonin N-domain and a 35-residue internal peptide in the Cdh23 cytoplasmic tail. Contrary to a previous model, we found no role for the Cdh23 C-terminal PDZ (PSD-95/Dlg/ZO-1)-binding motif and observed that Cdh23 bound similar levels of harmonin with or without the exon 68 peptide. We also examined two proteins involved in stereocilium elongation. The stereociliary actin-bundling protein espin was targeted to CL4 cell microvilli and caused microvillar elongation, whereas espin with the c.2469delGTCA or c.1988delAGAG human deafness mutation showed defects in microvillar targeting and elongation. The unconventional myosin motor myosin XVa accumulated at the tips of espin-elongated microvilli, by analogy to its location in stereocilia, whereas myosin XVa with the c.4351G>A or c.4669A>G human deafness mutation did not, revealing functional deficits in motor activity.",
author = "Lili Zheng and Jing Zheng and Whitlon, {Donna S} and Jaime Garcia-Anoveros and Bartles, {James Richard}",
year = "2010",
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T1 - Targeting of the hair cell proteins cadherin 23, harmonin, myosin XVa, espin, and prestin in an epithelial cell model

AU - Zheng, Lili

AU - Zheng, Jing

AU - Whitlon, Donna S

AU - Garcia-Anoveros, Jaime

AU - Bartles, James Richard

PY - 2010/5/26

Y1 - 2010/5/26

N2 - We have developed an advantageous epithelial cell transfection model for examining the targeting, interactions, and mutations of hair cell proteins. When expressed in LLC-PK1-CL4 epithelial cells (CL4 cells), the outer hair cell protein prestin showed faithful domain-specific targeting to the basolateral plasma membrane. We examined the consequences of mutations affecting prestin activity and assigned a targeting role to the cytoplasmic tail. The stereociliary link protein cadherin 23 (Cdh23) was targeted to the plasma membrane of CL4 cell microvilli, the topological equivalent of stereocilia. In cells coexpressing the Cdh23 cytoplasmic binding protein harmonin, a large fraction of harmonin became colocalized with Cdh23 in microvilli. Using this assay and in vitro protein binding assays, we formulated an alternative model for Cdh23-harmonin binding, in which the primary interaction is between the harmonin N-domain and a 35-residue internal peptide in the Cdh23 cytoplasmic tail. Contrary to a previous model, we found no role for the Cdh23 C-terminal PDZ (PSD-95/Dlg/ZO-1)-binding motif and observed that Cdh23 bound similar levels of harmonin with or without the exon 68 peptide. We also examined two proteins involved in stereocilium elongation. The stereociliary actin-bundling protein espin was targeted to CL4 cell microvilli and caused microvillar elongation, whereas espin with the c.2469delGTCA or c.1988delAGAG human deafness mutation showed defects in microvillar targeting and elongation. The unconventional myosin motor myosin XVa accumulated at the tips of espin-elongated microvilli, by analogy to its location in stereocilia, whereas myosin XVa with the c.4351G>A or c.4669A>G human deafness mutation did not, revealing functional deficits in motor activity.

AB - We have developed an advantageous epithelial cell transfection model for examining the targeting, interactions, and mutations of hair cell proteins. When expressed in LLC-PK1-CL4 epithelial cells (CL4 cells), the outer hair cell protein prestin showed faithful domain-specific targeting to the basolateral plasma membrane. We examined the consequences of mutations affecting prestin activity and assigned a targeting role to the cytoplasmic tail. The stereociliary link protein cadherin 23 (Cdh23) was targeted to the plasma membrane of CL4 cell microvilli, the topological equivalent of stereocilia. In cells coexpressing the Cdh23 cytoplasmic binding protein harmonin, a large fraction of harmonin became colocalized with Cdh23 in microvilli. Using this assay and in vitro protein binding assays, we formulated an alternative model for Cdh23-harmonin binding, in which the primary interaction is between the harmonin N-domain and a 35-residue internal peptide in the Cdh23 cytoplasmic tail. Contrary to a previous model, we found no role for the Cdh23 C-terminal PDZ (PSD-95/Dlg/ZO-1)-binding motif and observed that Cdh23 bound similar levels of harmonin with or without the exon 68 peptide. We also examined two proteins involved in stereocilium elongation. The stereociliary actin-bundling protein espin was targeted to CL4 cell microvilli and caused microvillar elongation, whereas espin with the c.2469delGTCA or c.1988delAGAG human deafness mutation showed defects in microvillar targeting and elongation. The unconventional myosin motor myosin XVa accumulated at the tips of espin-elongated microvilli, by analogy to its location in stereocilia, whereas myosin XVa with the c.4351G>A or c.4669A>G human deafness mutation did not, revealing functional deficits in motor activity.

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U2 - 10.1523/JNEUROSCI.0852-10.2010

DO - 10.1523/JNEUROSCI.0852-10.2010

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SP - 7187

EP - 7201

JO - Journal of Neuroscience

JF - Journal of Neuroscience

SN - 0270-6474

IS - 21

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