Abstract
BackgroundThe 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.ResultsFrom ≈ 3 × 106 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.ConclusionsThe 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.
Highlights
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
We have identified two novel extracellular matrix proteins latent transforming growth factor β-binding protein 3 and fibulin-1C that interact with the extracellular domain of proHB-EGF
Yeast two-hybrid screening of the monkey cDNA library using the extracellular domain of proHB-EGF as the bait fusion The initial screening of Ϸ 3 × 106 clones of the monkey cDNA library with the bait fusion of GAL4 binding domain fused to the extracellular domain of proHB-EGF yielded 294 colonies that were able to grow on Trp-HisLeu- media and that gave positive β-galactosidase activity in the β-galactosidase filter paper assay
Summary
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. CDNA clones containing nucleotide sequences encoding domains of two proteins were obtained and their derived amino acid sequences were evaluated. The precursor of heparin-binding EGF-like growth factor (proHB-EGF) is found on a wide variety of cell surfaces and is present in numerous tissue types [1]. In our laboratory, we cloned the cDNA encoding proHB-EGF from monkey Vero cells and identified this cell-surface molecule as a receptor for diphtheria toxin (DT) [2]. BMC Cell Biology 2002, 3 http://www.biomedcentral.com/1471-2121/3/2 to that of the cell surface-expressed precursor of human heparin-binding EGF-like growth factor (proHB-EGF) [3,4]. Membranebound proHB-EGF has juxtacrine growth factor activity [7] and acts as a DT receptor [2,8]
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