Targeting of the hair cell proteins cadherin 23, harmonin, myosin XVa, espin, and prestin in an epithelial cell model.
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.
Zheng
Lili
L
Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.
Zheng
Jing
J
Whitlon
Donna S
DS
García-Añoveros
Jaime
J
Bartles
James R
JR
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000070585:Cadherin Related Proteins; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D008954:Models, Biological; D016296:Mutagenesis / Q000235:genetics; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D000076988:Sulfate Transporters; D014162:Transfection / Q000379:methods
D048868:Adaptor Proteins, Signal Transducing; D027321:Anion Transport Proteins; C442480:CDH23 protein, human; D000070585:Cadherin Related Proteins; D015820:Cadherins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; C493274:ESPN protein, human; C485245:MYO15A protein, human; D008840:Microfilament Proteins; C498719:SLC26A5 protein, human; D000076988:Sulfate Transporters; C118970:USH1C protein, human; D049452:Green Fluorescent Proteins; D009218:Myosins