Latent transforming growth factor β-binding protein-3 and fibulin-1C interact with the extracellular domain of the heparin-binding EGF-like growth factor precursor

BACKGROUND: The membrane-bound cell-surface precursor and soluble forms of heparin-binding epidermal growth factor-like growth factor (HB-EGF) contribute to many cellular developmental processes. The widespread occurrence of HB-EGF in cell and tissue types has led to observations of its role in such cellular and tissue events as tumor formation, cell migration, extracellular matrix formation, wound healing, and cell adherence. Several studies have reported the involvement of such extracellular matrix proteins as latent transforming growth factor β-binding protein, TGF-β, and fibulin-1 in some of these processes. To determine whether HB-EGF interacts with extracellular matrix proteins we used the extracellular domain of proHB-EGF in a yeast two-hybrid system to screen a monkey kidney cDNA library. cDNA clones containing nucleotide sequences encoding domains of two proteins were obtained and their derived amino acid sequences were evaluated. RESULTS: From ≈ 3 × 10(6) screened monkey cDNA clones, cDNA clones were recovered that contained nucleotide sequences encoding domains of the monkey latent transforming growth factor-β binding protein-3 (MkLTBP-3) and fibulin-1C protein. The amino acid sequence derived from the MkLTBP-3 gene shared 98.6% identity with human LTBP-3 and 86.7% identity with mouse LTBP-3 amino acid sequences. The amino acid sequence derived from the monkey fibulin-1C gene shared 97.2% identity with human fibulin-1C. Yeast two-hybrid screens indicate that LTBP-3 and fibulin-1C interact with proHB-EGF through their calcium-binding EGF-like modules. CONCLUSIONS: The interactions of the extracellular domain of proHB-EGF with LTBP-3 and fibulin-1C suggest novel functions for HB-EGF between cell and tissue surfaces

Analysis of Thisbe and Pyramus functional domains reveals evidence for cleavage of Drosophila FGFs

Background: As important regulators of developmental and adult processes in metazoans, Fibroblast Growth Factor (FGF) proteins are potent signaling molecules whose activities must be tightly regulated. FGFs are known to play diverse roles in many processes, including mesoderm induction, branching morphogenesis, organ formation, wound healing and malignant transformation; yet much more remains to be learned about the mechanisms of regulation used to control FGF activity. Results: In this work, we conducted an analysis of the functional domains of two Drosophila proteins, Thisbe (Ths) and Pyramus (Pyr), which share homology with the FGF8 subfamily of ligands in vertebrates. Ths and Pyr proteins are secreted from Drosophila Schneider cells (S2) as smaller N-terminal fragments presumably as a result of intracellular proteolytic cleavage. Cleaved forms of Ths and Pyr can be detected in embryonic extracts as well. The FGF-domain is contained within the secreted ligand portion, and this domain alone is capable of functioning in the embryo when ectopically expressed. Through targeted ectopic expression experiments in which we assay the ability of full-length, truncated, and chimeric proteins to support cell differentiation, we find evidence that (1) the C-terminal domain of Pyr is retained inside the cell and does not seem to be required for receptor activation and (2) the C-terminal domain of Ths is secreted and, while also not required for receptor activation, this domain does plays a role in limiting the activity of Ths when present. Conclusions: We propose that differential protein processing may account for the previously observed inequalities in signaling capabilities between Ths and Pyr. While the regulatory mechanisms are likely complex, studies such as ours conducted in a tractable model system may be able to provide insights into how ligand processing regulates growth factor activity

Synthesis and subcellular fate of proteins encoded by the mouse int-2 (FGF3) gene

The int-2 gene encodes a member of the FGF family and was discovered as a proto-oncogene transcriptionally activated in tumours induced by Mouse Mammary Tumour Virus. Int-2 transcription is rarely detected in adult mouse tissues apart from low levels in brain and testis, but in situ hybridisation has revealed widespread expression during embryogenesis. The predicted int-2 gene product has a molecular mass of 27 kD and a hydrophobic sequence at the N-terminus which acts as a signal peptide for vectorial synthesis into the endoplasmic reticulum. The products have proved difficult to detect in natural sources known to contain int-2 RNA such as mammary tumours and embryo-derived cell lines. Consequently the protein has been characterised by expression of cloned cDNAs in COS-1 cells using an SV40-based plasmid vector, in insect cells infected with recombinant baculoviruses and by translation of synthetic sense RNA in cell-free systems supplemented with canine pancreatic microsomes. These studies identified four major int-2 products ranging in size from 27.5 kD to 31.5 kD which arise by post-translational modification of a 28.5 kD primary translation product. Although int-2 proteins are targeted to the secretory pathway, they have only been detected at very low levels in the medium and the associated extracellular matrix of transfected COS-1 cells. Cell-free translation systems programmed with synthetic int-2 RNA identified an additional N-terminally extended product which initiates from an in-frame CUG codon located upstream of the first AUG. Immunofluorescent staining of transfected COS-1 cells showed that a substantial proportion of this extended product localised in the cell nucleus, while a truncated int-2 protein lacking both the N-terminal extension and the signal peptide was exclusively nuclear. These observations suggested that the signals required for nuclear localisation of int-2 were encoded in the body of the molecule, but only functioned when entry into the secretory pathway was compromised. Two nuclear targeting sequences were identified by mutagenesis. Fusion of these motifs to the cytosolic protein pyruvate kinase did not result in effective nuclear localisation. However chimaeras containing int-2 and hst, an exclusively secreted member of the FGF-family, identified a targeting signal sequence with superficial resemblance to the bipartite nuclear localisation sequence of Xenopus nucleoplasmin. Initiation at alternative codons changes the sub-cellular fate of the protein and in principle provides a means imparting distinct functions upon the different int-2 products. Although a biological activity has yet to be assigned to the nuclear forms, the secreted protein is transforming in a NIH3T3 focus assay. The efficiency of focus formation was augmented by mutations which prevented initiation at the CUG codon or at an upstream AUG in the +1 reading frame, resulting in elevated levels of secreted int-2. Thus transformation was shown to require a high level of int-2 synthesis and secretion. In agreement with these findings, mutations which reduce the efficiency of secretion reduce the focus forming ability of NIH3T3 cells transfected with int-2 cDNAs, substantiating the notion that transformation is effected through an autocrine loop mechanism that involves a cell surface receptor

The plasmin-antiplasmin system: structural and functional aspects

The plasmin-antiplasmin system plays a key role in blood coagulation and fibrinolysis. Plasmin and α2-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into soluble fragments. However, besides plasmin(ogen) and α2-antiplasmin the system contains a series of specific activators and inhibitors. The main physiological activators of plasminogen are tissue-type plasminogen activator, which is mainly involved in the dissolution of the fibrin polymers by plasmin, and urokinase-type plasminogen activator, which is primarily responsible for the generation of plasmin activity in the intercellular space. Both activators are multidomain serine proteases. Besides the main physiological inhibitor α2-antiplasmin, the plasmin-antiplasmin system is also regulated by the general protease inhibitor α2-macroglobulin, a member of the protease inhibitor I39 family. The activity of the plasminogen activators is primarily regulated by the plasminogen activator inhibitors 1 and 2, members of the serine protease inhibitor superfamil

The Kinetics of the Hydrogen/Deuterium Exchange of Epidermal Growth Factor Receptor Ligands

Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 310-helix and random coil with varying contributions of β-sheets and β-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-α (TGF-α), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min−1 (Tyr), 0.09 min−1 (Arg, β-turns), and 1.88 × 10−3 min−1 (β-sheets and 310-helix); and for TGF-α 0.91 min−1 (Tyr), 0.27 min−1 (Arg, β-turns), and 1.41 × 10−4 min−1 (β-sheets). The time constants for AR 0.47 min−1 (Tyr), 0.04 min−1 (Arg), and 1.00 x 10−4 min−1 (buried 310-helix, β-turns, and β-sheets); for HB-EGF 0.89 min−1 (Tyr), 0.14 min−1 (Arg and 310-helix), and 1.00 x 10−3 min−1 (buried 310-helix, β-sheets, and β-turns); and for epiregulin 0.16 min−1 (Tyr), 0.03 min−1 (Arg), and 1.00 x 10−4 min−1 (310-helix and β-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state

Amphiregulin (areg) And Epidermal Growth Factor (egf): Disparate In Egfr Signaling And Trafficking

We have previously shown that SUM-149 human breast cancer cells require an AREG/EGFR autocrine loop for cell proliferation. We also demonstrated that AREG can increase EGFR stability and promote EGFR localization to the plasma membrane. In the presented dissertation we successfully knocked-down AREG expression in SUM-149 cells by lenti-viral infection of AREG shRNA. In the absence of AREG expression, SUM-149 cell growth was slowed, but not completely inhibited. Furthermore, cells infected with AREG shRNA constructs showed an increase in EGFR protein expression by western blot. Immunofluorescence and confocal microscopy showed that following AREG knock-down, EGFR continued to localize to the cell surface. Soft agar assays demonstrated that AREG knock-down cells retain anchorage-independent growth capacity. Additionally mammosphere forming assays and Adefluor staining analysis showed that knock-down of AREG expression did not affect the expression of stem cell 158 phenotypes. However, following AREG knock-down, SUM-149 cells demonstrated a dramatic decrease in their ability to invade a Matrigel matrix. Consistent with this observation, microarray analysis comparing cells infected with a non-silencing vector to the AREG knock-down cells, identified genes associated with the invasive phenotype such as RHOB and DKK1, and networks associated with cell motility such as integrinlinked kinase signaling, and focal adhesion kinase signaling. AREG was also found to modulate WNT and Notch signaling in SUM-149 cells. In an additional microarray study, changes in gene expression were analyzed from cDNA transcribed from RNA isolated from MCF10A cells growing in the presence of AREG or EGF and after 24 hours withdrawl of the respective ligand. Genes regulating WNT signaling, but not NOTCH signaling, were altered in the MCF10A cells. Thus, the pathway that AREG/EGFR signaling effects is contextually dependent on the cell type that it is functioning in. We conclude that AREG functions in regulating the invasive phenotype, and we propose that this regulation may be through altered signaling that occurs when AREG activates plasma membrane localized EGFR