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Exp Cell Res ; 314(11-12): 2199-211, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18570918

RESUMO

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Animais , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Endossomos/metabolismo , Células Epiteliais/citologia , Humanos , Iodetos/metabolismo , Transporte Proteico/fisiologia , Proteínas Qa-SNARE/genética , Proteínas Qb-SNARE/genética , Proteínas R-SNARE/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas SNARE/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
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