EH Domain-containing Protein Research Articles

Identification of proteins differentially expressed between capillary endothelial cells of hepatocellular carcinoma and normal liver in an orthotopic rat tumor model using 2-D DIGE

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with few treatment options. It is a hypervascular tumor in which angiogenesis plays a critical role in its progression. Tumor capillary endothelial cells (TECs) in HCC are known to originate from liver sinusoid endothelial cells, which then go through a capillarization process to become morphologically as well as functionally different TECs. In this work, we investigated proteins differentially expressed between freshly isolated TECs and sinusoid endothelial cells from well-formed rat HCC using 2-D DIGE coupled with MALDI-TOF/TOF MS. Thirty-eight unique proteins were identified to be differentially expressed more than twofold between the two endothelial cell types. Amongst the differentially expressed proteins, two novel endothelial markers, EH domain-containing protein 3 and galectin-3, were confirmed by Western blot and immunohistochemistry in both rat and human HCC samples. We showed that EH domain-containing protein 3 is significantly down-regulated in TECs, but galectin-3 is up-regulated. We propose possible roles of these two proteins in tumor vessel development in HCC.

Endocytosis Function of a Ligand-Gated Ion Channel Homolog in Caenorhabditis elegans

Ligand-gated ion channels are transmembrane proteins that respond to a variety of transmitters, including acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate [1 and 2]. These proteins play key roles in neurotransmission and are typically found in the nervous system and at neuromuscular junctions [3]. Recently, acetylcholine receptor family members also have been found in nonneuronal cells, including macrophages [4], keratinocytes [5], bronchial epithelial cells [5], and endothelial cells of arteries [6]. The function of these channels in nonneuronal cells in mammals remains to be elucidated, though it has been shown that the acetylcholine receptor alpha7 subunit is required for acetylcholine-mediated inhibition of tumor necrosis factor release by activated macrophages [4]. We show that cup-4, a gene required for efficient endocytosis of fluids by C. elegans coelomocytes, encodes a protein that is homologous to ligand-gated ion channels, with the highest degree of similarity to nicotinic acetylcholine receptors. Worms lacking CUP-4 have reduced phosphatidylinositol 4,5-bisphosphate levels at the plasma membrane, suggesting that CUP-4 regulates endocytosis through modulation of phospholipase C activity.

REPS2/POB1 is downregulated during human prostate cancer progression and inhibits growth factor signalling in prostate cancer cells.

During progression of prostate cancer, cellular changes occur, leading to a transition from androgen-dependent to androgen-independent growth. One aspect of this transition is a switch from androgens to growth factors, like epidermal growth factor (EGF), as primary regulators of proliferation. We examined the involvement of REPS2/POB1 in this process. REPS2/POB1 is an EH domain-containing protein, reported to be involved in signalling via RalBP1 and to play a role in endocytosis of EGF receptors. Furthermore, the protein is relatively highly expressed in androgen-dependent as compared to androgen-independent human prostate cancer cell lines and xenografts. Next to the known REPS2/POB1 protein, an open reading frame encoding REPS2/POB1, with 139 additional amino-acid residues at the NH(2)-terminus, was cloned and found to be expressed in prostate cancer cells. Overexpression, by transient transfection, of both forms of REPS2/POB1 in prostate cancer cell lines, induced apoptosis within 48 h. At shorter time intervals after transfection, signalling towards a TPA response element luciferase reporter was found to be inhibited. From these experiments, it is concluded that REPS2/POB1, through its influence on the Ral signalling pathway, is involved in growth factor signalling. Decreased expression of REPS2/POB1 during progression of prostate cancer may therefore result in loss of control of growth factor signalling and consequently in loss of control of cell proliferation.

Characterization of EHD4, an EH domain-containing protein expressed in the extracellular matrix.

To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human placenta library was used and a new extracellular protein discovered. The cDNA sequence of the new protein encodes 541 amino acid residues. This cDNA sequence is identical to EHD4, a recently described member of the EH domain family of proteins. Two mRNAs of 4.4 and 3.0 kilobases were present in human skin fibroblasts and most tissues tested but were most prevalent in the heart. The chromosomal localization of the gene for this new protein was determined to be at 15q14-q15. Three polyclonal peptide antibodies were made against synthetic EHD4 peptides. The affinity-purified antibodies were used in immunofluorescent staining of developing limbs and matrices produced by human skin fibroblasts and mouse NIH3T3 fibroblasts in culture. Embryonic rat limb cartilage was strongly stained throughout development, and cultured fibroblasts deposited an extracellular filamentous network containing EHD4. In non-denaturing extracts of fetal bovine cartilage and in human skin fibroblast culture media, two components of approximately 220 and 158 kDa were observed, which, after reduction, migrated as a 56-kDa component on SDS-polyacrylamide gel electrophoresis. EHD4 is the first extracellular matrix protein described that contains an EH domain.

Intersectin Can Regulate the Ras/MAP Kinase Pathway Independent of Its Role in Endocytosis

We previously identified intersectin, a multiple EH and SH3 domain-containing protein, as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. More recently, we identified mammalian Sos, a guanine-nucleotide exchange factor for Ras, as an intersectin SH3 domain-binding partner. We now demonstrate that overexpression of intersectin's SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4 degrees C or with phenylarsine oxide, treatments previously established to inhibit EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation.