EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.
The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.SH3 domains bind to proline-rich peptides that fold into a polyproline type 2 helix. Many SH3-binding proteins contain relatively long proline-rich domains (PRD) 1 with multiple potential SH3 interaction sites (1-4). Given the relatively low specificity of peptide recognition mediated by SH3 domains, it is not clear whether all these interactions, which are identified in vitro, are of functional significance. A second question that arises is whether SH3 domains bind rather unspecifically to many sites along the PRD or rather form specific complexes by binding to unique and distinct sites.Dynamin, synaptojanin, and synapsin, three proteins that are concentrated in the pre-synaptic region of nerve terminals, bear proline-rich regions that bind to diverse SH3-containing proteins. Synapsin I is the main synaptic ligand of the SH3 domain of the adapter protein Grb2 in vitro (2). Recently it has been reported that the same proline-rich D region of synapsin I interacts with c-Src and stimulates its tyrosine kinase activity (5). The physiological significance of these interactions is not clear yet. In contrast, strong evidence supports the notion that disruption of the interaction between amphiphysin and the PRD of dynamin impairs synaptic vesicle endocytosis (6). Dynamin is a GTPase that forms a collar at the neck of forming endocytic vesicles and participates in the fission process that results in the formation of free vesicles (7). Several other SH3-containing proteins have been shown to bind to dynamin in vitro (1, 8 -10).Syna...
Screening cDNA libraries from solid human tumors with sera of autologous patients (SEREX) has proven to be a powerful approach to identifying tumor antigens recognized by the humoral arm of the immune system. In many cases, application of this methodology has led to the discovery of novel tumor antigens as unknown gene products. We tried to improve the potency of the SEREX approach by combining it with phage-display technology. We designed a new lambda vector to express protein fragments as N-terminal fusions to the D capsid protein and generated high-complexity cDNA libraries from human breast carcinoma cell lines and solid tumors. Screening these phage-displayed libraries required limited amounts of sera from patients and efficiently identified several tumor antigens specifically reacting with sera from breast cancer patients. © 2003 Wiley-Liss, Inc. Key words: breast cancer; immune response; phage displayTumor-associated antigens recognized by humoral effectors of the immune system are an attractive target for diagnostic and therapeutic approaches to human cancer. A considerable number of this type of antigen have been identified over recent years through screening expression cDNA libraries from human solid tumors with sera of the autologous patients (SEREX, serologic identification of antigens by recombinant expression cloning). [1][2][3][4] This type of screening of a cDNA expression library by conventional methods requires the preparation of a large number of membrane filters blotted with bacteriophage plaques that are then searched with a specific probe. In the case of the SEREX experiments, the screening is performed using sera from cancer patients, which are usually available in very limited quantity. The second limitation is that such immunoscreening procedure does not allow selection of antigens that are recognized by sera from different patients.We tried to overcome these limitations and to simplify the screening procedure by performing affinity selection of cDNA libraries in very small volumes by combining the SEREX approach with that of phage-display technology. Phage-display technology is based on the insertion of foreign nucleotide sequences into genes encoding for various coat proteins of filamentous phage, resulting in a heterogeneous mixture of phages, each displaying the different peptide sequence encoded by a corresponding insert. A physical link between a displayed fusion protein and DNA encoded for it make this phage target selectable.Phage-display technology was introduced in 1985 by Smith 5 and has been widely used since for generating and screening peptide libraries to identify ligands for various kinds of receptor molecules. 6 -8 However, significantly less striking progress has been reported on the use of filamentous phage as display vector for constructing cDNA libraries. In most cases, foreign peptides are displayed on the filamentous phage capsid as N-terminal fusions to the major (pVIII) or the minor (pIII) coat protein. Large protein domains fused to pVIII disturb (with some rare...
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