Protein: DP03364 (UniProt Q15582)
Region: residues 641	to 645
Annotation: localization (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
The RGD motif present in the carboxy-terminus of TGFBI is necessary, but not sufficient, for SKOV3 cell adhesion and is dispensable for adhesion of ovarian cancer cells lacking ß3 integrin expression.
Both TGFBI and periostin contain conserved motifs shown to mediate binding to the integrin receptor family. However, although TGFBI and periostin retain the four conserved fasciclin I domains, periostin contains a longer carboxy-terminus lacking an RGD motif, which is present in TGFBI (Figure 1A). Importantly, the RGD
motif has been implicated in integrin receptor binding and has been shown to be necessary for cell adhesion to various extracellular proteins, including fibronectin [36].
Both TGFBI and periostin contain conserved Fasciclin I and EMI domains, while only TGFBI contains an RGD motif.
Unlike periostin, the carboxy-terminus of rTGFBI supports adhesion of ovarian cancer cells and is dependent on an intact RGD motif
The carboxyterminus of TGFBI (aa 498–683), which contains the fourth fasciclin I domain and the RGD motif, was capable of supporting SKOV3 cell adhesion similar to fulllength rTGFBI. However, the fourth fasciclin I domain alone (aa 498–637), previously shown to support HUVEC and human fibroblast cell adhesion [15,43], and the central domain (aa 24–506) were unable to support SKOV3 adhesion (Figure 5B, 5C). Furthermore, mutagenesis of the RGD motif to amino acid residues RAE in the carboxy-terminal truncated form of TGFBI (aa 498–683) abrogated adhesion of SKOV3 cells (Figure 5B, 5C).
To further understand how the fourth fasciclin I domain and the RGD motif cooperate with other TGFBI domains, we evaluated whether mutation of the RGD motif to amino acid residues RAE would affect the ability of full-length TGFBI to support SKOV3 adhesion. In these experiments we found that the RGD to RAE mutation in full-length TGFBI significantly reduced SKOV3 adhesion (Figure 6A).
Therefore, we tested whether the ERGDEL peptide derived from TGFBI was capable of competitively inhibiting adhesion of ovarian cancer cells to fibronectin and rTGFBI. Pretreatment of cells with the classical fibronectin
GRGDSP peptide was capable of inhibiting adhesion to both fibronectin and rTGFBI (Figure 6B). By contrast, pretreatment with the TGFBI ERGDEL peptide did not alter adherence to fibronectin and rTGFBI (Figure 6B). Therefore, the RGD motif of TGFBI is necessary, but is not sufficient, to support adhesion of SKOV3 cells and
binding either requires a greater number of flanking amino acids or a complex with the fourth Fasciclin I domain.
Our results suggest that the RGD motif of full-length TGFBI is necessary, but not sufficient, for ovarian cancer cell adhesion, thus indicating it may cooperate with flanking residues or other motifs, potentially present within the fourth Fasciclin I domain to mediate this process. Importantly, we found that the TGFBI derived RGD peptide (ERGDEL) was unable to competitively inhibit SKOV3 adhesion to rTGFBI, suggesting its use as a therapeutic agent to inhibit TGFBI function may depend on the cellular context.
RGD motif (642-644) with flanking residues.