The GGAs are a multidomain protein family implicated in protein trafficking between the Golgi and endosomes. Here, the VHS domain of GGA2 was shown to bind to the acidic cluster-dileucine motif in the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor (CI-MPR). Receptors with mutations in this motif were defective in lysosomal enzyme sorting. The hinge domain of GGA2 bound clathrin, suggesting that GGA2 could be a link between cargo molecules and clathrin-coated vesicle assembly. Thus, GGA2 binding to the CI-MPR is important for lysosomal enzyme targeting.

The VHS (Vps-27, Hrs and STAM) domain is a 140 residue long domain present in the very NH 2 -terminus of at least 60 proteins. Based on their functional characteristics and on recent data on the involvement of VHS in cargo recognition in trans-Golgi, VHS domains are considered to have a general membrane targeting/cargo recognition role in vesicular trafficking. Structurally, VHS is a right-handed superhelix of eight helices with charged surface patches probably serving as sites of protein^protein recognition and docking. ß

The molecular basis of the selectivity and the details of the vesicle formation in endocytic and secretory pathways are still poorly known and most probably involve as yet unidentified components. Here we describe the cloning, expression, and tissue and cell distribution of a novel protein of 67 kDa (called Vear) that bears homology to several endocytosis-associated proteins in that it has a VHS domain in its N terminus. It is also similar to ␥-adaptin, the heavy subunit of AP-1, in having in its C terminus a typical "ear" domain. In immunofluorescence microscopy, Vear was seen in the Golgi complex as judged by a typical distribution pattern, a distinct colocalization with the Golgi marker ␥-adaptin, and a sensitivity to treatment of cells with brefeldin A. In cell fractionation, Vear partitioned with the post-nuclear membrane fraction. In transfection experiments, hemagglutinin-tagged full-length Vear and truncated Vear lacking the VHS domain assembled on and caused compaction of the Golgi complex. Golgi association without compaction was seen with the ear domain of Vear, whereas the VHS domain alone showed a diffuse membrane-and vesicle-associated distribution. The Golgi association and the bipartite structure along with the differential targeting of its domains suggest that Vear is involved in heterotypic vesicle/suborganelle interactions associated with the Golgi complex. Tissue-specific function of Vear is suggested by its high level of expression in kidney, muscle, and heart.

We describe the cloning and characterization of a new cytoplasmic protein designated epidermal growth factor receptor-associated protein with SH3-and TAM domains (EAST). It contains an Src homology 3 domain in its midregion and a tyrosine-based activation motif in its COOH terminus. Antibodies to EAST recognize a 68-kDa protein that is present in most chicken tissues. An epidermal growth factor (EGF)-dependent association between the EGF receptor (EGFR) and EAST was shown by reciprocal immunoprecipitation/immunoblotting studies with specific antibodies. Activated EGFR catalyzed the tyrosine phosphorylation of EAST, as judged by an in vitro kinase assay with both immunoprecipitated and purified EGFR. Immunoprecipitation/immunoblotting experiments also demonstrated an association between EAST and eps15, an EGFR substrate associated with clathrin-coated pits and vesicles, which is essential in the endocytotic pathway. The association between EAST and eps15 was not affected by EGF treatment. In immunofluorescence microscopy, EAST was shown to partially colocalize with clathrin. The sequence of the NH 2 -terminal portion of EAST shows a high degree of similarity with a group of proteins involved in endocytosis or vesicle trafficking. Thus, EAST is a novel signal transduction component probably involved in EGF signaling and in the endocytotic machinery.Signal transduction proteins are characterized by their capacity to specifically associate with other proteins to form multimolecular assemblies (1). In the case of receptor tyrosine kinases and receptor tyrosine kinase-induced intracellular signaling, such protein interactions are mediated by distinct protein domains. Among these, the Src homology 2 (SH2) 1 domain, which binds Tyr(P) residues in a specific context, and the Src homology 3 (SH3) domain, which binds sequences characterized by polyproline tracts, are the best known (2). In the epidermal growth factor receptor (EGFR), for instance, binding of the epidermal growth factor (EGF) leads to the phosphorylation of multiple tyrosine residues by the kinase activity of the receptor. These Tyr(P) residues serve as docking sites for various downstream, SH2-containing, signaling elements (3). These, in turn, can associate with other signaling proteins or substrates via other binding modules, such as SH3 domains.As new proteins and interactions are being discovered, new motifs are also disclosed. Thus, the phosphotyrosine-binding domain present in Shc and IRS-1, for example, recognizes Tyr(P) in a manner different from SH2 domains (4). Similarly, a distinct new type of tyrosine-containing, SH2-binding domain, termed tyrosine-based activation motif (TAM), has been found in antigen receptor molecules (5). TAM-containing receptors lack an intrinsic kinase activity and use TAM motifs to recruit and activate nonreceptor protein tyrosine kinases, such as members of the Src and Syk families (6), as their effectors.Many of the known signaling pathways are still only partially characterized, and their regulation is poorly underst...

The relationship between glutathione deficiency, glycogen metabolism and ascorbate synthesis was investigated in isolated murine hepatocytes. Glutathione deficiency caused by various agents increased ascorbate synthesis with a stimulation of glycogen breakdown. Increased ascorbate synthesis from UDP-glucose or gulonolactone could not be further affected by glutathione depletion. Fructose prevented the stimulated glycogenolysis and ascorbate synthesis caused by glutathione consumption. Reduction of oxidised glutathione by dithiothreitol decreased the elevated glycogenolysis and ascorbate synthesis in diamide or menadione treated hepatocytes. Our results suggest that a change in GSI-I/GSSG ratio seems to be a sufficient precondition of altering glycogenolysis and a consequent ascorbate synthesis.