EH domain containing genes and proteins

Details EH domain containing genes and proteins EH domain containing genes and proteins

1999-05-17

2003-04-22

Fredman, Jeffrey (Department: 1637)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024300, C435S006120, C435S091200

Reexamination Certificate

active

06552177

ABSTRACT:

FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to eps15 homology (EH) domain containing proteins, to polynucleotide sequences encoding said EH domain containing proteins, to oligonucleotides and oligonucleotide analogs derived from said polynucleotide sequences, to a display library displaying short peptides derived from said EH domain containing proteins, to antibodies recognizing said EH domain containing proteins, to peptides or peptide analogs derived from said EH domain containing proteins, and to pharmaceutical compositions and methods of employing said peptides or peptide analogs, said oligonucleotides and oligonucleotide analogs, and/or said polynucleotide sequences to up-regulate or down-regulate clathrin coated pit mediated endocytosis and thereby insulin growth factor 1 receptor (IGF1 receptor) signaling.
Citation or identification of any reference in this section or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
Abbreviations used herein include AP—adaptor protein complex; BBS—Bardet-Biedl Syndrome; ECL—enhanced chemiluminescence; EGFR—epidermal growth factor receptor; EH—eps15 homology; Eps—epidermal growth factor receptor-pathway-substrate; EST—expressed sequence tag; GFP—green fluorescent protein; HRP—horseradish peroxidase; IGF1—insulin-like growth factor 1; ocd—osteochondrodystrophy; ORF—open reading frame; PBS—phosphate buffered saline.
The diverse effects growth factors have on cell proliferation, differentiation and metabolism are mediated by interaction with cell surface receptors. There are several receptor families which convey their ligand-induced signals through different intracellular mechanisms. One family of receptors posses tyrosine kinase activity (Darnell et al., 1995) and includes the EGF receptor, insulin receptor and the IGF1 receptor. Binding of these receptors to their ligands induces cascade of events, leading to sequestration of the ligand bound receptor in endocytic vesicles (Kirchhausen et al., 1997; Mukherjee et al., 1997; Warren et al., 1998). This process depends on the specific interaction of clathrin and the clathrin adaptor protein complex, AP-2, with specific accessory factors. It has been shown that the EGFR phosphorylates at least two proteins, eps15 and eps15R, after which each one of them may interact, through accessory proteins, with AP-2.(Benmerah et al., 1998; Coda et al., 1998). This interaction leads to endocytosis of the ligand bound EGFR. Pan1p, the yeast homologue of eps15, was also shown to function as a multivalent adaptor that coordinates protein—protein interactions essential for endocytosis (Wendland and Emr, 1998).
Ligand induced endocytosis is a regulated process, leading to the formation of clathrin coated pits containing the ligand bound receptor. The spontaneous polymerization of clathrin triskelions is thought to cause the pits to expand and eventually to form the clathrin coated vesicle. These vesicles loose their coat after endocytosis, forming the early endosome. Endocytosed receptors, after their dissociation from the ligand due to the low pH in the early endosome, are usually recycled to the plasma membrane or are destined to the lysosomes, were they are degraded. For EGF receptor, the phosphorylation of eps15 leads, most probably, to binding of at least another specific protein, epsin,(Chen et al., 1998) and this protein complex seems to recruit AP-2. (Iannolo et al., 1997)., which then binds to clathrin.
Eps15 and eps15R contain three domains: One is an N-terminal domain containing three repeats of the EH motif, directing protein—protein interactions through the amino acids NPF (asparagine-proline-phenylalanine, SEQ ID NO: 11) of target proteins. The EH domain spans about 100 amino acids, about 50% of which are conserved between different proteins containing this domain. EH domains are frequently present in multiple copies and might also include EF-hand calcium binding motifs. It has been shown that the second EH domain consists of a pair of EF hand motifs, the second of which binds tightly to Ca
2+
. The NPF containing motif binds in a hydrophobic pocket of the EH domain, between two alpha helices, and the binding is mediated by a critical aromatic interaction (Benmerah et al., 1998; de Beer et al., 1998; Di Fiore et al., 1997; Fazioli et al., 1993; Schumacher et al., 1995; Tebar et al., 1997). A second domain contains heptad repeats, characteristic of coiled-coil structure, which directs dimerization (and most probably oligomerization). The third domain is C-terminal region and has a proline-rich region and a repeated DPF (aspartic acid-proline-phenylalanine, SEQ ID NO:12) motif.(Benmerah et al., 1998; Di Fiore et al., 1997; Fazioli et al., 1993; Schumacher et al., 1995; Tebar et al., 1997).
There is a growing number of EH-containing proteins, like intersectin .(Yamabhai et al., 1998) Ese1 and Ese2 (Sengar et al., 1999) and they all seem to be associated with the intracellular routing machinery (Di Fiore et al., 1997). Another EH containing protein is the Drosophila Dap160, a neural specific protein that anchors proteins required for endocytosis. (Roos and Kelly, 1998). It has been suggested that endocytosis is only one of several intracellular activities in which EH containing proteins participate and may also regulate (Coda et al., 1998).
Several proteins, containing NPF motifs, have been identified as interacting with eps15 and participating in clathrin coated pit mediated endocytosis, like: epsin (Chen et al., 1998) and dynamin,(Roos and Kelly, 1998).
While reducing the present invention to practice, two new and highly homologous genes isolated from both human and mouse, named EHD 1 (which is referred to in U.S. Pat. No. 09/026,898 as testiline and has the yet unpublished accession Nos. AF099011 for the human cDNA and AF099186 for the mouse cDNA) and EHD2 were cloned, sequenced, mapped, their expression characterized and function analyzed. The proteins encoded by the isolated genes contain an EH domain which, as described above, is known to modulate interactions with endocytic vesicles. Based on the pattern of the EHD1 and EHD2 genes expression and on their interaction with other cellular proteins it is concluded that the protein products of these genes participate in clathrin coated pit mediated endocytosis of IGF1 receptor, following its binding to its ligand.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided an isolated nucleic acid comprising a genomic, complementary or composite polynucleotide sequence encoding a polypeptide having an N-terminal region containing a nucleotide binding consensus site, a central coiled-coil structure and a C-terminal region including an eps15 homology (EH) domain. According to a preferred embodiment the polypeptide encoded by the polynucleotide participates in endocytosis processes.
The polynucleotide according to this aspect of the present invention preferably encodes a polypeptide which is at least 75% homologous to SEQ ID NOs:4, 5, 9 or 10 as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap creation penalty equals 8 and gap extension penalty equals 2.
According to preferred embodiments, the polynucleotide according to this aspect of the present invention encodes a polypeptide as set forth in SEQ ID NOs:4, 5, 9 or 10 or a portion thereof, preferably a portion which retains EHD1 or 2 activity.
Alternatively or additionally, the polynucleotide according to this aspect of the present invention is preferably hybridizable with SEQ ID NOs:1, 2, 3, 6, 7 or 8.
Hybridization for long nucleic acids (e.g., above 200 bp in length) is effected according to preferred embodiments of the present invention by stringent or moderate hybridization, wherein stringent hybridization is effected by a hybridization solution containing 10% dextrane sulfate, 1 M NaCl, 1% SDS and 5×10
6
cpm
32
p labeled probe, at 65° C.,

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