RESUMO
The Dishevelled, EGL-10 and pleckstrin (DEP) domain is a globular protein domain that is present in about ten human protein families with well-defined structural features. A picture is emerging that DEP domains mainly function in the spatial and temporal control of diverse signal transduction events by recruiting proteins to the plasma membrane. DEP domains can interact with various partners at the membrane, including phospholipids and membrane receptors, and their binding is subject to regulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosfoproteínas/metabolismo , Proteínas RGS/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Proteínas Desgrenhadas , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas RGS/química , Proteínas RGS/genéticaRESUMO
Epac1 is a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap. Upon cAMP binding, Epac1 undergoes a conformational change that results in its release from autoinhibition. In addition, cAMP induces the translocation of Epac1 from the cytosol to the plasma membrane. This relocalization of Epac1 is required for efficient activation of plasma membrane-located Rap and for cAMP-induced cell adhesion. This translocation requires the Dishevelled, Egl-10, Pleckstrin (DEP) domain, but the molecular entity that serves as the plasma membrane anchor and the possible mechanism of regulated binding remains elusive. Here we show that Epac1 binds directly to phosphatidic acid. Similar to the cAMP-induced Epac1 translocation, this binding is regulated by cAMP and requires the DEP domain. Furthermore, depletion of phosphatidic acid by inhibition of phospholipase D1 prevents cAMP-induced translocation of Epac1 as well as the subsequent activation of Rap at the plasma membrane. Finally, mutation of a single basic residue within a polybasic stretch of the DEP domain, which abolishes translocation, also prevents binding to phosphatidic acid. From these results we conclude that cAMP induces a conformational change in Epac1 that enables DEP domain-mediated binding to phosphatidic acid, resulting in the tethering of Epac1 at the plasma membrane and subsequent activation of Rap.
Assuntos
Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ácidos Fosfatídicos/química , Fatores de Troca do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Lipídeos/química , Lipossomos/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Proteínas rap1 de Ligação ao GTP/químicaRESUMO
PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.