Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins. Many such motifs conform to the consensus YXX⌽, where ⌽ represents a bulky hydrophobic residue. This motif interacts with the medium chain ( ) subunits of adaptor complexes that link the cytosolic domains of integral membrane proteins to the clathrin coat involved in vesicle formation. The YXX⌽ motif is similar to motifs in which the tyrosine residue is phosphorylated by tyrosine kinases. Tyrphostins (structural analogs of tyrosine) are inhibitors of tyrosine kinases and function by binding to the active sites of the enzymes. We previously showed that, in vitro and in yeast two-hybrid interaction assays, some tyrphostins can inhibit the interaction between YXX⌽ motifs and the 2 subunit of the AP-2 adaptor complex (Crump, C., Williams, J. L., Stephens, D. J., and Banting, G. (1998) J. Biol. Chem. 273, 28073-28077). A23 is such a tyrphostin. We now show that molecular modeling of tyrphostin A23 into the tyrosinebinding pocket in 2 provides a structural explanation for A23 being able to inhibit the interaction between YXX⌽ motifs and 2. Furthermore, we show that A23 inhibited the internalization of 125 I-transferrin in Heb7a cells without having any discernible effect on the morphology of compartments of the endocytic pathway. Control tyrphostins, active as inhibitors of tyrosine kinase activity, but incapable of inhibiting the YXX⌽ motif/ 2 interaction, did not inhibit endocytosis. These data are consistent with A23 inhibition of the YXX⌽ motif/ 2 interaction in intact cells and with the possibility that different tyrphostins may be used to inhibit specific membrane trafficking events in eukaryotic cells.The trafficking and intracellular targeting of many transmembrane proteins depend on motifs found within their cytosolic domains. One such motif contains a critical tyrosine residue within the sequence YXX⌽, where ⌽ represents a bulky hydrophobic residue (1, 2). Tyrosine-based motifs conforming to this consensus sequence can interact directly with the medium chain ( ) subunits of heterotetrameric adaptor complexes involved in several different intracellular trafficking pathways. The AP-2 adaptor complex facilitates incorporation of transmembrane proteins containing YXX⌽ motifs into clathrincoated vesicles at the cell surface (1, 3-9), whereas AP-1, AP-3, and AP-4 adaptor complexes are involved in other intracellular vesicular transport steps (reviewed in Refs. 10 -13). The subunits from all four adaptor complexes have been shown to interact with the YXX⌽ motif, with the precise sequence and context of this motif determining the specificity of the interaction (14, 15). Mutation of residues within and around the YXX⌽ motif affect interaction with subunits; but, in all cases, mutation of the tyrosine residue in the motif (e.g. to alanine) blocks interaction with the subunit (4, 6, 8). Structural studies on peptides containing these motifs have generated data that hav...

SummaryEnteropathogenic Escherichia coli (EPEC) are a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli encode a type III secretion system (TTSS) to transfer effector proteins into host cells, a process which is essential for virulence. In addition to generation of A/E lesions, the TTSS is also implicated in the ability of EPEC to invade cultured cells but the effector proteins responsible for promoting invasion have not been identified. In this paper we confirm the requirement of TTSS in EPEC invasion and demonstrate important roles for the Map and Tir effector molecules. Whereas in trans expression of Tir in the tir mutant restored invasion to wild-type levels, similar complementation of the map mutation by in trans expression of Map results in a hyperinvasive phenotype. The Map effector protein has two distinct functions within host cells, mediating Cdc42-dependent filopodia formation and targeting mitochondria to elicit dysfunction. The former function appears to be related to Map's ability to promote invasion as this was inhibited by interference with Cdc42 signalling. Conversely, Map targeting to mitochondria is not necessary for invasion. Promotion of EPEC invasion by Tir appears to involve interaction with intimin but is independent of pedestal formation, and intimin-Tir interaction is neither necessary nor sufficient for invasion. Comparison of the invasiveness of strains lacking Tir and/or Map with wild-type or mutant strains expressing the effectors in trans provides evidence that Map and Tir stimulate invasion by synergistic mechanisms. This synergism, which is in stark contrast to the antagonistic actions of Map and Tir in regulating filopodia and pedestal formation, further illustrates the complex interplay between EPEC effectors.

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins. Many such motifs conform to the consensus YXX+(+ represents a bulky hydrophobic residue). This motif interacts with the medium chain (mu) subunits of adaptor complexes which link the cytosolic domain of the integral membrane protein to the clathrin coat involved in vesicle formation. The motif YXX+ is similar to motifs which are phosphorylated by tyrosine kinases. Tyrphostins (structural analogues of tyrosine) are inhibitors of tyrosine kinases: can they inhibit interactions between mu chains and YXX+ motifs? We have investigated several tyrphostins and found that, in vitro and in the yeast two hybrid system, some are capable of inhibiting the intraction between YXX+ motifs and different medium chains. We have also shown that tyrphostins which block these interactions affect the internalisation and recyling of the transferrin receptor in live cells.At present little is known regarding the mechanism of metabotopic glutamate receptor (mGluR) trafficking. In order to study this, we inserted a hemagglutinin (HA) epitope tag between residues 57-58 of the extracellular domain of the rat mGluRla splice variant. In HEK293 cells transiently transfected with HA-mGlula, the epitope tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (1OpM; 30 min) the HA-mGluRla internalized to early endosomes and an endocytic recycling compartment. Further quantification of receptor endocytosis was provided by ELISA experiments which showed that the HA-mGluRla underwent rapid agonist-(glutamate 10 inpM) induced internalization with a t o,5 of 19 ? 3 min. To determine whether agonist-induced mGluRla internalization is an arrestin-and dynamin-dependent process cells were cotransfected with HA-mGluRla and either dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant construct with HA-mGluRla strongly inhibited glutmate-induced (10 pM; 30 min) internalization. In addition arrestin-2-GFP or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluRla. Taken together our observations demonstrate that agonist-induced internalization of mGluRla is an arrestin-and dynamin-dependent process.Arrestin-and Dynamin-dependent. 73The effect of pertussis toxin on the internalisation of the Somatostatin agonists are rapidly and efficiently endocytosed with the somatostatin2 receptor. The aim of this study was to determine the role of G protein coupling on internalisation by examining the effect of pertussis toxin treatment. The accumulation of internalised agonists and receptors is governed by a number of processes including hormone-receptor association, dissociation, receptor endocytosis and recycling back to the plasma membrane. We have established a mathematical model describing these processes and used this model in the mechanistic interpretat...

Skin barrier function is conferred by the outer layer of epidermis, the stratum corneum, and is essential for terrestrial life. Quantitative trans-epidermal water loss assays show that barrier forms late in embryogenesis, permitting the foetus to survive a terrestrial environment at birth. Using qualitative in situ assays for skin permeability, we show that barrier forms in a patterned manner late in mouse gestation. Barrier forms at specific epidermal sites, then spreads around the embryo as a moving front. The moving front of permeability change is accompanied by multiple changes in the outer, stratum corneum-precursor cells. We use the permeability assays to show that final stages of cornified envelope assembly are coordinated with initial stages of barrier formation. Hence the whole-mount permeability assays record developmental acquisition of a known, essential component of the adult barrier. We demonstrate the authenticity of the whole-mount assays after maternal glucocorticoid therapy (known to accelerate barrier formation) and in additional species including the rat where barrier formation is well characterized by TEWL assay (Aszterbaum, M., Menon, G. K., Feingold, K. R. and Williams, M. L. Pediatr. Res. 31, 308–317). The demonstration of patterned barrier formation in other species suggests patterned change as the universal mode of embryonic barrier acquisition. These results highlight the importance of patterning as a mode of epidermal maturation during development.