The Pathway Interaction Database (PID, http://pid.nci.nih.gov) is a freely available collection of curated and peer-reviewed pathways composed of human molecular signaling and regulatory events and key cellular processes. Created in a collaboration between the US National Cancer Institute and Nature Publishing Group, the database serves as a research tool for the cancer research community and others interested in cellular pathways, such as neuroscientists, developmental biologists and immunologists. PID offers a range of search features to facilitate pathway exploration. Users can browse the predefined set of pathways or create interaction network maps centered on a single molecule or cellular process of interest. In addition, the batch query tool allows users to upload long list(s) of molecules, such as those derived from microarray experiments, and either overlay these molecules onto predefined pathways or visualize the complete molecular connectivity map. Users can also download molecule lists, citation lists and complete database content in extensible markup language (XML) and Biological Pathways Exchange (BioPAX) Level 2 format. The database is updated with new pathway content every month and supplemented by specially commissioned articles on the practical uses of other relevant online tools.

The Biomolecular Interaction Network Database (BIND) (http://bind.ca) archives biomolecular interaction, reaction, complex and pathway information. Our aim is to curate the details about molecular interactions that arise from published experimental research and to provide this information, as well as tools to enable data analysis, freely to researchers worldwide. BIND data are curated into a comprehensive machinereadable archive of computable information and provides users with methods to discover interactions and molecular mechanisms. BIND has worked to develop new methods for visualization that amplify the underlying annotation of genes and proteins to facilitate the study of molecular interaction networks. BIND has maintained an open database policy since its inception in 1999. Data growth has proceeded at a tremendous rate, approaching over 100 000 records. New services provided include a new BIND Query and Submission interface, a Standard Object Access Protocol service and the Small Molecule Interaction Database (http://smid.blueprint.org) that allows users to determine probable small molecule binding sites of new sequences and examine conserved binding residues. INTRODUCTIONIn light of the vast scientific resources made available through genomics, the science of deciphering molecular mechanisms is expanding rapidly. Scientists who once hunted for disease genes or sought to distinguish key concepts in evolution are now turning their attention to the details of molecular assembly and mechanism to further understand medicine and the key concepts underlying biology. The Biomolecular Interaction Network Database (BIND) was designed to store complete information about molecular assembly through a database structure in order to archive interactions and reactions arising from biopolymers (protein, RNA and DNA), as well as small molecules, lipids and carbohydrates. Detailed information about molecular mechanism, such as the chemical product(s) of an enzymatic reaction, can be encoded in BIND. The underlying ontology of the BIND database is chemistry, and as such, BIND is capable of storing information about molecular interactions to atomic resolution. The taxonomic scope of BIND is

The Pathway Interaction Database (PID, "http://pid.nci.nih.gov":http://pid.nci.nih.gov) is a freely available collection of curated and peer-reviewed pathways composed of human molecular signaling and regulatory events and key cellular processes. Created in a collaboration between the U.S. National Cancer Institute and Nature Publishing Group, the database serves as a research tool for the cancer research community and others interested in cellular pathways, such as neuroscientists, developmental biologists, and immunologists. PID offers a range of search features to facilitate pathway exploration. Users can browse the predefined set of pathways or create interaction network maps centered on a single molecule or cellular process of interest. In addition, the batch query tool allows users to upload long list(s) of molecules, such as those derived from microarray experiments, and either overlay these molecules onto predefined pathways or visualize the complete molecular connectivity map. Users can also download molecule lists, citation lists and complete database content in extensible markup language (XML) and Biological Pathways Exchange (BioPAX) Level 2 format. The database is updated with new pathway content every month and supplemented by specially commissioned articles on the practical uses of other relevant online tools.

RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1-DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4alpha and RasGRP4beta. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4beta do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4beta C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.

RasGRP1 is a Ras-activating exchange factor that is positively regulated by translocation to membranes. RasGRP1 contains a diacylglycerol-binding C1 domain, and it has been assumed that this domain is entirely responsible for RasGRP1 translocation. We found that the C1 domain can contribute to plasma membrane-targeted translocation of RasGRP1 induced by ligation of the B cell antigen receptor (BCR). However, this reflects cooperativity of the C1 domain with the previously unrecognized Plasma membrane Targeter (PT) domain, which is sufficient and essential for plasma membrane targeting of RasGRP1. The adjacent suppressor of PT (SuPT) domain attenuates the plasma membranetargeting activity of the PT domain, thus preventing constitutive plasma membrane localization of RasGRP1. By binding to diacylglycerol generated by BCR-coupled phospholipase C␥2, the C1 domain counteracts the SuPT domain and enables efficient RasGRP1 translocation to the plasma membrane. In fibroblasts, the PT domain is inactive as a plasma membrane targeter, and the C1 domain specifies constitutive targeting of RasGRP1 to internal membranes where it can be activated and trigger oncogenic transformation. Selective use of the C1, PT, and SuPT domains may contribute to the differential targeting of RasGRP1 to the plasma membrane versus internal membranes, which has been observed in lymphocytes and other cell types. INTRODUCTIONRasGRP1 is a guanine nucleotide exchange factor that couples antigen receptors to the activation of Ras GTPases (Dower et al., 2000;Ebinu et al., 2000;Priatel et al., 2002;Bivona et al., 2003;Caloca et al., 2003b;Layer et al., 2003;Norment et al., 2003;Oh-hora et al., 2003;Guilbault and Kay, 2004;Perez de Castro et al., 2004;Reynolds et al., 2004;Zugaza et al., 2004;Coughlin et al., 2005;Roose et al., 2005). Deletion of the RasGRP1 gene perturbs the immunological selection and activation of lymphocytes (Dower et al., 2000;Priatel et al., 2002Priatel et al., , 2006Layer et al., 2003) and mast cells (Liu et al., 2007), whereas aberrant expression of RasGRP1 initiates oncogenic transformation of lymphocytes (Li et al., 1999;Mikkers et al., 2002;Suzuki et al., 2002;Kim et al., 2003;Dupuy et al., 2005;Klinger et al., 2005), fibroblasts (Ebinu et al., 1998;Tognon et al., 1998) and keratinocytes (Oki-Idouchi and Lorenzo, 2007).To be active as an exchange factor, RasGRP1 must be localized to cell membranes where Ras GTPases reside. This requirement provides an opportunity for positive or negative regulation. RasGRP1 contains a C1 domain that binds the lipid second messenger diacylglycerol (DAG), or DAGmimetic phorbol esters (Ebinu et al., 1998;Lorenzo et al., 2000;Shao et al., 2001;Rong et al., 2002;Carrasco and Merida, 2004;Madani et al., 2004). Treatment of cells with DAG or phorbol esters results in the translocation of RasGRP1 to membranes (Ebinu et al., 1998;Tognon et al., 1998;Bivona et al., 2003;Rambaratsingh et al., 2003;Caloca et al., 2004;Stone et al., 2004), and it stimulates Ras activation via RasGRP1 (Ebinu et al., 1998;...

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The Pathway Interaction Database (PID, http://pid.nci.nih.gov) is a freely available collection of curated and peer-reviewed signaling pathways composed of human biomolecular interactions and cellular processes. Created in a collaboration between the U.S. National Cancer Institute and Nature Publishing Group, the database is a research tool for cell biologists, biochemists, computational biologists and bioinformaticians. The PID offers a range of tools to facilitate pathway exploration. Users can browse the pre-defi ned set of pathways and also create interaction network maps centered on a single molecule of interest or an extensive list of molecules. In addition, users can download complete data sets in extensible markup language (XML) and Biological Pathway Exchange (BioPAX) Level 2 formats. The database is updated every month and supplemented by a concise editorial section that provides synopses of recent noteworthy papers in cell signaling and specially commissioned articles on the practical uses of other relevant online tools. Users can sign up for free email alerts or RSS feeds to receive database updates. Curation principles• Human model system: We focus on human data. Interactions in other mammals that are inferred to occur in humans may be included with appropriate evidence codes.• Biological relevance: Meaningful networks of undisputed interactions are synthesized into pathways. Pathways selected for curation are based on suggestions made by our users, potential drugs targets and other biomolecules we know to be of interest to researchers.• Authority: Molecular interactions are identifi ed in primary peer-reviewed literature. Editors judge whether an interaction is physiologically relevant and assign evidence codes to each interaction. All pathways are reviewed by experts in the fi eld for accuracy and completeness.

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