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

Abstract

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 beta-binding protein, TGF-beta, 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 approximately equal to 3 x 10(6) screened monkey cDNA clones, cDNA clones were recovered that contained nucleotide sequences encoding domains of the monkey latent transforming growth factor-beta 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.

Figure 1

Diagrammatic representation of the human LTBP-3 protein. Note that for the amino acid sequence derived for the monkey LTBP-3 (Mk LTBP-3), the monkey, mouse and human LTBP-3 proteins have the same structural features. Symbols represent structural features of the protein: cysteine-rich regions, patterned octagons; proline/glycine rich region, solid line; fibrillin motif, open rectangle; calcium-binding type II EGF-like modules, open octagons; cysteine-rich TGF-bp repeats, dotted punched tape symbol; TGF-bp-like repeat, wavy lined punched tape symbol. The numbers 1-5 represent different domains of the protein. The construct insert of pGAD424/LTBP-3 EGF #1-8, containing the region of Mk LTBP-3 that interacts with the extracellular domain of proHB-EGF (DTREx), is shown as a forward slashed box.

Figure 2

Overlapping monkey cDNA clones containing monkey LTBP-3 sequence. The numbers displayed next to each clone represent the nucleotides of the cDNA clone. The numbering system is based on that presented for the mouse LTBP-3 cDNA sequence in which the A of the intitiator Met codon is nucleotide 1 (Yin et al. 1995). The fusion proteins encoded by each of the cDNA clones demonstrated interaction with the extracellular domain of proHB-EGF when tested for β-galactosidase activity using the yeast two-hybrid system assay.

Figure 3

Diagrammatic representation of the human fibulin-1C protein (modified after Argraves et al. [33]). Note that for the derived amino acid sequence obtained for the monkey fibulin-1C protein, the human and monkey fibulin-1C proteins have identical structural features. Symbols represent the following structural features: anaphylatoxin type I repeats, diamonds; potential N-glycosylation site, small circle; EGF-adjoining sequence, dotted oval; calcium-binding type II EGF-like modules, open octagons; potential Asn/Asp hydroxylation site, triangles, potential o-glycosylation site, inverted triangles; fibulin-type module, large hexagon. The construct insert of pGAD424/Fib-1C EGF #1–5 is represented by a dotted box. The construct insert of pGAD424/Fib-1C EGF #5-COOH, containing a region of fibulin-1C that interacts with the extracellular domain of monkey proHB-EGF (DTREx), is represented by a forward slashed box.