To study the relationship of IA-2 antibodies (IA-2A) to other autoantibodies and genetic risk markers in insulin-dependent diabetes mellitus (IDDM), 758 children and adolescents younger than 15 years of age (mean age 8.4 years) with newly diagnosed diabetes were analysed for IA-2A, GAD antibodies (GADA) and insulin autoantibodies (IAA) with radiobinding assays, for islet cell antibodies (ICA) with immunofluorescence and for HLA DR alleles by serology. IA-2A were detected in 85.9% of cases with no association with gender or age. An overwhelming majority of the patients (71.3%) tested positive for three or more antibodies, and 90.7% for at least two. Fifty-four subjects (7.1%) had one antibody detectable, whereas only 2.1% of the patients tested negative for all four. A higher proportion of patients was positive for IA-2A and/or GADA than for ICA alone (95.5 vs 84.2%, p < 0.001). The prevalence and level of IA-2A were increased in cases carrying HLA DR4/non-DR3 compared with other DR combinations. The results indicate that almost all patients with newly diagnosed childhood IDDM can be identified by screening with these four autoantibodies. The combination of IA-2A and/or GADA had a higher sensitivity for IDDM than ICA alone. The close association between IA-2A and HLA DR4, the strongest single allele predisposing to IDDM, suggests that IA-2A may be a more specific marker of beta-cell destruction than GADA, which have been shown to associate with the DR3 allele and thyroid autoimmunity.

The amiloride‐sensitive Na+ channel constitutes the rate‐limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha‐spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride‐sensitive) Na+ channel (alpha ENaC) binds to alpha‐spectrin in vivo, as determined by co‐immunoprecipitation. This binding is mediated by the SH3 domain of alpha‐spectrin which binds to a unique proline‐rich sequence within the C‐terminal region of alpha rENaC. Accordingly, the C‐terminal region is sufficient to mediate binding to intact alpha‐spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C‐terminal proline‐rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha‐spectrin. In contrast, microinjected fusion protein containing the N‐terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells.

Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes.

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...

Src-homology 3 (SH3) domain is a 60 -70-amino acid motif present in a large variety of signal transduction and cytoskeletal proteins. We used reverse transcriptase-polymerase chain reaction with degenerate and specific primers and chicken brain mRNA to clone a cDNA that codes for a novel SH3 domain-containing protein. The sequence predicts a 448-amino acid polypeptide with a molecular mass of 51,971 daltons. In the amino terminus, it shows a very high propensity for ␣-helicity, suggesting coiled-coil and possibly a higher order oligomeric arrangement. In the carboxyl terminus, there is a unique SH3 sequence. In Northern blotting, a major 3.7-kilobase and a minor 7.2-kilobase transcript was detected in most chicken tissues. In immunofluorescence microscopy and immunoelectron microscopy on cultured chicken fibroblasts, the protein was localized to focal adhesions in which it showed a distinct codistribution with the focal adhesion proteins vinculin, talin, and paxillin. Phosphoamino acid analysis showed that in cultured chicken heart fibroblasts, the protein contains phosphoserine, but no phosphothreonine or phosphotyrosine, and that the phosphorylation is not dependent on fibronectin. We propose this protein the name FAP52, for Focal Adhesion Protein of 52 kDa, and suggest that it forms part of the multimolecular complex constituting focal adhesion sites.