Epsin and AP180/CALM are important endocytic accessory proteins that are believed to be involved in the formation of clathrin coats. Both proteins associate with phosphorylated membrane inositol lipids through their epsin N-terminal homology domains and with other components of the endocytic machinery through short peptide motifs in their carboxyl-terminal segments. Using hydrodynamic and spectroscopic methods, we demonstrate that the parts of epsin 1 and AP180 that are involved in protein-protein interactions behave as poorly structured flexible polypeptide chains with little or no conventional secondary structure. The predominant cytosolic forms of both proteins are monomers. Furthermore, we show that recombinant epsin 1, like AP180, drives in vitro assembly of clathrin cages. We conclude that the epsin N-terminal homology domaincontaining proteins AP180/CALM and epsin 1 have a very similar molecular architecture that is designed for the rapid and efficient recruitment of the principal coat components clathrin and AP-2 at the sites of coated pit assembly.Clathrin-coated vesicles are involved in a number of membrane transport processes, including receptor-mediated endocytosis, recycling of synaptic vesicles, and sorting of lysosomal enzymes (1). Despite our detailed knowledge of the structural components of the coat and the identity of many endocytic accessory proteins, the molecular events leading to coat formation on the plasma membrane remain elusive. Among the factors considered to be important for coat formation are the neuronal proteins AP180, its ubiquitously expressed homolog CALM (clathrin assembly lymphoid myeloid leukemia protein), and epsin 1 (2, 3). All three proteins have a globular epsin N-terminal homology (ENTH) 1 domain in common that is constructed from 8 to 10 ␣-helical rods (4 -6). This domain mediates binding to the rare membrane lipid phosphatidylinositol 4,5-bisphosphate (PI-4,5-P 2 ), which is generally regarded as a major recruiter of components for the endocytic machinery to the plasma membrane (7,8). AP180 and epsin 1 interact directly with clathrin and the ␣-and ␤-appendage domains of the AP-2 adaptor complex (3, 9 -14). AP180 binding to clathrin promotes assembly of clathrin triskelia into a population of small cages with a narrow size distribution (15, 16). In neuronal tissue of Drosophila, a knockout of the AP180 ortholog LAP reduces the number of clathrin-coated vesicles; moreover, their size range is much wider than in wild-type flies (17). These studies have led to the suggestion that AP180 and its orthologs might be involved in the control of vesicle size and thus support the notion of playing an important role in vesicle formation. More recently, the potential of AP180 to recruit clathrin to lipid surfaces and to assemble it there was demonstrated with PI-4,5-P 2 -containing liposomes and lipid monolayers (7). When a combination of AP-2 and AP180 was added to the monolayer, the clathrin lattices became deeply invaginated (7). Whereas the ENTH domains of AP180 and epsin 1 app...

SummaryIntercellular adhesion molecule-1 (ICAM-1, CD54) is a ligand for the integrins lymphocyte function associated-1 (LFA-1, CD11a/CD18) and complement receptor-3 (Mac-l, CD11b/ CD 18) making it an important participant in many immune and inflammatory processes. Modified recombinant soluble ICAM-1 formed dimers. This result indicated that the ectodomain of ICAM-1 contains homophilic interaction sites. Soluble ICAM-1 dimers bind to solid-phase purified LFA-I with high avidity (dissociation constant [Ka] = 8 nM) in contrast to soluble ICAM-1 monomers whose binding was not measurable. Cell surface ICAM-1 was found to be dimeric based on two distinct criteria. First, a monoclonal antibody specific for monomeric soluble ICAM-1, CA7, binds normal ICAM-1 poorly at the cell surface; this antibody, however, binds strongly to two mutant forms of [CAM-1 when expressed at the cell surface, thus identifying elements required for dimer formation. Second, chemical cross-linking of cell surface ICAM-1 on transfected cells and tumor necrosis factor-activated endothelial cells results in conversion of a portion oflCAM-1 to a covalent dimer. Cell surface ICAM-1 dimers are more potent ligands for LFA-l-dependent adhesion than ICAM-1 monomers. While many extracellular matrix-associated ligands of integrins are multimeric, this is the first evidence of specific, functionally important homodimerization of a cell surface integrin ligand.

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We evaluated whether a probiotic supplementation in dogs with food responsive diarrhoea (FRD) has beneficial effects on intestinal cytokine patterns and on microbiota. Twenty-one client-owned dogs with FRD were presented for clinically needed duodeno- and colonoscopy and were enrolled in a prospective placebo (PL)-controlled probiotic trial. Intestinal tissue samples and faeces were collected during endoscopy. Intestinal mRNA abundance of interleukin (IL)-5, -10, -12p40 and -13, tumour necrosis factor-alpha, transforming growth factor-beta1 and interferon (IFN)-gamma were analysed and numbers of Lactobacillus spp., Bifidobacterium spp., Enterococcus spp. and Enterobacteriaceae and supplemented probiotic bacteria were determined in faeces. The Canine Inflammatory Bowel Disease Activity Index, a scoring system comprising general attitude, appetite, faecal consistency, defecation frequency, and vomitus, decreased in all dogs (p < 0.0001). Duodenal IL-10 mRNA levels decreased (p = 0.1) and colonic IFN-gamma mRNA levels increased (p = 0.08) after probiotic treatment. Numbers of Enterobacteriaceae decreased in FRD dogs receiving probiotic cocktail (FRD(PC)) and FRD dogs fed PL (FRD(PL)) during treatment (p < 0.05), numbers of Lactobacillus spp. increased in FRD(PC after) when compared with FRD(PC before) (p < 0.1). One strain of PC was detected in five of eight FRD(PC) dogs after probiotic supplementation. In conclusion, all dogs clinically improved after treatment, but cytokine patterns were not associated with the clinical features irrespective of the dietary supplementation.

The I domain of lymphocyte function–associated antigen (LFA)-1 contains an intercellular adhesion molecule (ICAM)-1 and ICAM-3 binding site, but the relationship of this site to regulated adhesion is unknown. To study the adhesive properties of the LFA-1 I domain, we stably expressed a GPI-anchored form of this I domain (I-GPI) on the surface of baby hamster kidney cells. I-GPI cells bound soluble ICAM-1 (sICAM-1) with a low avidity and affinity. Flow cell experiments demonstrated a specific rolling interaction of I-GPI cells on bilayers containing purified full length ICAM-1 or ICAM-3. The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1–containing membranes. In contrast, the interaction of I-GPI cells with ICAM-3 was blocked by MEM-83. Rolling of I-GPI cells was dependent on the presence of Mg2+. Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity. This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.