Acommon focus among molecular and cellular biologists is the identification of proteins that interact with each other. Yeast two-hybrid, cDNA expression library screening, and coimmunoprecipitation experiments are powerful methods for identifying novel proteins that bind to one's favorite protein for the purpose of learning more regarding its cellular function. These same techniques, coupled with truncation and mutagenesis experiments, have been used to define the region of interaction between pairs of proteins. One conclusion from this work is that many interactions occur over short regions, often less than 10 amino acids in length within one protein. For example, mapping studies and 3-dimensional analyses of antigen-antibody interactions have revealed that epitopes are typically 4-7 residues long (1). Other examples include protein-interaction modules, such as Src homology (SH) 2 and 3 domains, phosphotyrosine binding domains (PTB), postsynaptic density/disc-large/ZO1 (PDZ) domains, WW domains, Eps15 homology (EH) domains, and 14-3-3 proteins that typically recognize linear regions of 3-9 amino acids. Each of these domains has been the subject of recent reviews published elsewhere (2 3 4 5 6 7). Among the primary structures of many ligands for protein-protein interactions, the amino acid proline is critical. In particular, SH3, WW, and several new protein-interaction domains prefer ligand sequences that are proline-rich. In addition, even though ligands for EH domains and 14-3-3 domains are not proline-rich, they do include a single proline residue. This review highlights the analysis of those protein-protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.-Kay, B. K., Williamson, M. P., Sudol, M. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX 1-2 NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.The EH 1 domain has recently been described as a protein interaction module involved in endocytosis (1). The domain was first discovered in Eps15, a tyrosine kinase phosphorylation substrate of the epidermal growth factor receptor (2). Eps15 is a ϳ145,000 Da protein with three EH repeats and has been shown to be a component of endocytic vesicle intermediates (3-5). Biochemical analysis of the Eps15 EH domains have shown that they are likely involved in protein-protein interactions: far-Western blotting and affinity chromatography experiments demonstrate that a number of cellular proteins can bind to the Eps15 EH fusion protein (2), and recently, several potential cellular ligands have been identified (6). Within the Saccharomyces cerevisiae genome there are five EH domaincontaining proteins, two of which, Pan1 and End3, have been shown to have roles in endocytosis (7-9). Another protein interaction module is the Src homology 3 (SH3) domain. This domain is 50 -70 amino acids long and is present in numerous signal transduction and cytoskeletal proteins (10, 11). Examination of the ligand specificity of SH3 domains has revealed that they recognize proline-rich sequences containing the core peptide sequence PXXP (12, 13). SH3 domains have proposed roles in directing the assembly of NADPH oxidase subunits (14), modulating the activity of phosphatidylinositol 3Ј-kinase (15) and the GTPase activity of dynamin (16), as well as lo...

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These (14) used a synthetic peptide library to identify Src and P13K SH3 ligands with the consensus sequence RXLPPZP (Z = L for Src and R for P13K) and demonstrated that each SH3 domain bound its respective ligand with higher affinity than did the other.In an effort to develop a more detailed understanding of the specificity of SH3-ligand interactions, we have constructed a phage-displayed library (termed the PXXP library) encoding peptides of the form X6PXXPX6, where X represents any of the 20 naturally occurring amino acids and P represents invariant proline residues. Using this library, we have identified peptide ligands for SH3 domains of Src, Yes, Abl, Cortactin, p53bp2, PLCy, Crk, and Grb2. Each SH3 domain selects a set of peptide ligands sharing a distinct consensus motif; these motifs reflect the unique ligand preferences of each SH3 domain. MATERIALS AND METHODSPreparation of Glutathione S-Transferase (GST)-SH3 Fusion Proteins. Constructs encoding GST fusions to the Grb2 N-terminal (Grb2 N, aa 1-58), Grb2 C-terminal (Grb2 C, aa 154-217), Nck N-terminal (Nck N, aa 1-68), Nck middle (Nck M, aa 101-166), Nck C-terminal (Nck C, aa 191-257), p53bp2 (aa 454-530), or Src (aa 87-143) SH3 domains were generated by PCR cloning of the appropriate cDNAs into pGEX-2T (Pharmacia). The integrity of the constructs was confirmed by DNA sequencing. pGEX-derived constructs expressing GST fusions to the SH3 domains of Yes, Cortactin, Crk, Abl, and PLC,y were kindly provided by M. Sudol (Rockefeller University), J. T. Parsons (University of Virginia), M. Matsuda

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Based on the prevalence of modular protein domains, such as Src homology domain 3 and 2 (SH3 and SH2), among important signaling molecules, we have sought to identify new SH3 domain-containing proteins. However, modest sequence similarity among these domains restricts the use of DNA-based methods for this purpose. To circumvent this limitation, we have developed a functional screen that permits the rapid cloning of modular domains based on their ligand-binding activity. Using operationally defined SH3 ligands from combinatorial peptide libraries, we screened a series of mouse and human cDNA expression libraries. We found that 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, 10 of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome analysis and in bringing new targets into drug discovery programs.

Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrincoated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways.