Abstract
Del1 is a novel extracellular matrix protein encoding three Notch-like epidermal growth factor repeats, an RGD motif, and two discoidin domains. Del1 is expressed in an endothelial cell-restricted pattern during early development. In studies reported here, recombinant baculovirus Del1 protein was shown to promote alphavbeta3-dependent endothelial cell attachment and migration. Attachment of endothelial cells to Del1 was associated with clustering of alphavbeta3, the formation of focal complexes, and recruitment of talin and vinculin into these complexes. These events were shown to be associated with phosphorylation of proteins in the focal complexes, including the time-dependent phosphorylation of p125(FAK), MAPK, and Shc. When recombinant Del1 was evaluated in an in ovo chick chorioallantoic membrane assay, it was found to have potent angiogenic activity. This angiogenic activity was inhibited by a monoclonal antibody directed against alphavbeta3, and an RAD mutant Del1 protein was inactive. Thus Del1 provides a unique autocrine angiogenic pathway for the embryonic endothelium, and this function is mediated in part by productive ligation of integrin alphavbeta3.
Highlights
Del1 is a novel extracellular matrix protein encoding three Notch-like epidermal growth factor repeats, an RGD motif, and two discoidin domains
Preliminary data suggested that Del1 MJR is a matrix protein, and that the RGD motif could mediate endothelial cell attachment through integrin ligation
Recombinant Del1 Mediates Endothelial Cell Attachment in an RGD-dependent Manner—Recombinant Del1 MJR, Del1 MNR, and Del1 RAD proteins were expressed in Sf9 insect cells, purified on a nickel column, and fractions were analyzed by SDS-PAGE and Western blotting with polyclonal Del1 antisera
Summary
Del is a novel extracellular matrix protein encoding three Notch-like epidermal growth factor repeats, an RGD motif, and two discoidin domains. Attachment of endothelial cells to Del was associated with clustering of ␣v3, the formation of focal complexes, and recruitment of talin and vinculin into these complexes These events were shown to be associated with phosphorylation of proteins in the focal complexes, including the time-dependent phosphorylation of p125FAK, MAPK, and Shc. When recombinant Del was evaluated in an in ovo chick chorioallantoic membrane assay, it was found to have potent angiogenic activity. In vitro and in vivo functional studies suggested that Del may have a complex role in the regulation of vascular formation, serving to inhibit endothelial cell differentiation or contribute to the complex process of vascular remodeling Such a functional role was not reconciled with the molecular interaction of Del with the ␣v3 integrin receptor. Cell attachment assays revealed that Del could mediate endothelial cell attachment at concentrations similar to that observed with known matrix proteins such as vitronectin (VN) and fibronectin (FN), and
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