EHD4 and CDH23 are interacting partners in cochlear hair cells.
Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner.
Sengupta
Soma
S
Department of Communication Sciences and Disorders, Hugh Knowles Center, Northwestern University, Evanston, Illinois 60208, USA.
George
Manju
M
Miller
Katharine K
KK
Naik
Khurram
K
Chou
Jonathan
J
Cheatham
Mary Ann
MA
Dallos
Peter
P
Naramura
Mayumi
M
Band
Hamid
H
Zheng
Jing
J
D000818:Animals; D015820:Cadherins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003051:Cochlea / Q000166:cytology / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006198:Hair Cells, Auditory / Q000166:cytology / Q000378:metabolism; D006309:Hearing; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D015820:Cadherins; C499091:Cdh23 protein, mouse; D004268:DNA-Binding Proteins; C543675:EHD4 protein, mouse; C485640:Ehd1 protein, mouse; D009687:Nuclear Proteins; D033921:Vesicular Transport Proteins