RESUMEN
Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. Epsin 1 and 2 are most similar in their NH(2)-terminal region, which represents a module (epsin NH(2) terminal homology domain, ENTH domain) found in a variety of other proteins of the data base. The multiple DPW motifs, typical of the central region of epsin 1, are only partially conserved in epsin 2. Both proteins, however, interact through this central region with the clathrin adaptor AP-2. In addition, we show here that both epsin 1 and 2 interact with clathrin. The three NPF motifs of the COOH-terminal region of epsin 1 are conserved in the corresponding region of epsin 2, consistent with the binding of both proteins to Eps15. Epsin 2, like epsin 1, is enriched in brain, is present in a brain-derived clathrin-coated vesicle fraction, is concentrated in the peri-Golgi region and at the cell periphery of transfected cells, and partially colocalizes with clathrin. High overexpression of green fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.
Asunto(s)
Proteínas Portadoras/genética , Clatrina/metabolismo , Neuropéptidos/genética , Proteínas de Transporte Vesicular , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Vesículas Cubiertas/metabolismo , Cricetinae , ADN Complementario/química , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Neuropéptidos/química , Neuropéptidos/metabolismo , Fosfoproteínas/metabolismo , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Synaptojanin is a nerve terminal protein of relative molecular mass 145,000 which appears to participate with dynamin in synaptic vesicle recycling. The central region of synaptojanin defines it as a member of the inositol-5-phosphatase family, which includes the product of the gene that is defective in the oculocerebrorenal syndrome of Lowe. Synaptojanin has 5-phosphatase activity and its amino-terminal domain is homologous with the yeast protein Sac1 (Rsd1), which is genetically implicated in phospholipid metabolism and in the function of the actin cytoskeleton. The carboxy terminus, which is of different lengths in adult and developing neurons owing to the alternative use of two termination sites, is proline-rich, consistent with the reported interaction of synaptojanin with the SH3 domains of Grb2 (refs 1, 2). Synaptojanin is the only other major brain protein besides dynamin that binds the SH3 domain of amphiphysin, a presynaptic protein with a putative function in endocytosis. Our results suggest a link between phosphoinositide metabolism and synaptic vesicle recycling.