ABSTRACT
The development of inducible and conditional technologies allowed us to generate transgenic mouse models that faithfully recapitulate human tumorigenesis. It is possible to control, in time and space, the development of tumors in almost every mouse tissue. The result is that now we have available mouse models for all major human cancers. Novel noninvasive approaches to tumor imaging will enable us to follow tumor development and metastasis in vivo, as well as the effects of candidate therapeutic drugs. Such new generation tumor models, which accurately emulate the disease state in situ, should provide a useful platform with which to experimentally test drugs targeted to specific gene products, or combinations of genes that control rate-limiting steps of tumor development. In this review, we focus on the different mouse models for colon cancer.
Subject(s)
Disease Models, Animal , Neoplasms/genetics , Neoplasms/pathology , Adenomatous Polyposis Coli/genetics , Adenomatous Polyposis Coli/pathology , Animals , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Drug Evaluation, Preclinical , Humans , MiceABSTRACT
The Eps homology (EH) domain is a recently described protein binding module that is found, in multiple or single copies, in several proteins in species as diverse as human and yeast. In this work, we have investigated the molecular details of recognition specificity mediated by this domain family by characterizing the peptide-binding preference of 11 different EH domains from mammal and yeast proteins. Ten of the eleven EH domains could bind at least some peptides containing an Asn-Pro-Phe (NPF) motif. By contrast, the first EH domain of End3p preferentially binds peptides containing an His-Thr/Ser-Phe (HT/SF) motif. Domains that have a low affinity for the majority of NPF peptides reveal some affinity for a third class of peptides that contains two consecutive amino acids with aromatic side chains (FW or WW). This is the case for the third EH domain of Eps15 and for the two N-terminal domains of YBL47c. The consensus sequences derived from the peptides selected from phage-displayed peptide libraries allows for grouping of EH domains into families that are characterized by different NPF-context preference. Finally, comparison of the primary sequence of EH domains with similar or divergent specificity identifies a residue at position +3 following a conserved tryptophan, whose chemical characteristics modulate binding preference.
Subject(s)
Calcium-Binding Proteins/metabolism , Phosphoproteins/metabolism , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Calcium-Binding Proteins/chemistry , DNA Primers , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Peptides/metabolism , Phosphoproteins/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
During endocytosis, clathrin and the clathrin adaptor protein AP-2, assisted by a variety of accessory factors, help to generate an invaginated bud at the cell membrane. One of these factors is Eps15, a clathrin-coat-associated protein that binds the alpha-adaptin subunit of AP-2. Here we investigate the function of Eps15 by characterizing an important binding partner for its region containing EH domains; this protein, epsin, is closely related to the Xenopus mitotic phosphoprotein MP90 and has a ubiquitous tissue distribution. It is concentrated together with Eps15 in presynaptic nerve terminals, which are sites specialized for the clathrin-mediated endocytosis of synaptic vesicles. The central region of epsin binds AP-2 and its carboxy-terminal region binds Eps15. Epsin is associated with clathrin coats in situ, can be co-precipitated with AP-2 and Eps15 from brain extracts, but does not co-purify with clathrin coat components in a clathrin-coated vesicle fraction. When epsin function is disrupted, clathrin-mediated endocytosis is blocked. We propose that epsin may participate, together with Eps15, in the molecular rearrangement of the clathrin coats that are required for coated-pit invagination and vesicle fission.
Subject(s)
Calcium-Binding Proteins/physiology , Carrier Proteins/physiology , Clathrin/physiology , Endocytosis/physiology , Neuropeptides/physiology , Phosphoproteins/physiology , Vesicular Transport Proteins , Adaptor Protein Complex alpha Subunits , Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Animals , Blotting, Northern , Brain/metabolism , CHO Cells , Calcium-Binding Proteins/metabolism , Carrier Proteins/chemistry , Cricetinae , Membrane Proteins/metabolism , Molecular Sequence Data , Neuropeptides/chemistry , Phosphoproteins/metabolism , Protein Binding , Rats , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
eps15 and eps1SR are substrates of the epidermal growth factor (EGF) receptor kinase that are characterized by the presence of a protein:protein interaction domain, the EH domain, and by their ability to bind to the clathrin adaptor protein complex adaptor protein 2. Indirect evidence suggests that eps15 and eps15R are involved in endocytosis. Here we show that microinjection of antibodies against eps15 and eps15R inhibits internalization of EGF and transferrin. In addition, fragments of eps15 (encompassing its EH domains or the COOH-terminal region that binds to adaptor protein 2) inhibit EGF internalization or endocytosis of Sindbis virus. These results demonstrate that eps15 and eps15R are essential components of the endocytic machinery.
