Key events in tumor cell metastasis involves attachment, plasmin-media proteolysis, and migration in the extracellular matrix (ECM). Vitronec (VN) is a major adhesion cell protein in plasma and in the ECM. VN forms a complex with plasminogen activator inhibitor type-1 stabilizing PAI-I from rapid inactivation, and thereby modulating proteolysis in the ECM. At least three distinct cell surface receptors for VN, belonging to the integrin superfamily, have been identified in normal and tumor cells. The ligand-receptor interaction is mediated in part by the RGD sequence. Most cell adhesion ligands contain a RGD sequence, yet the receptors binding these ligands do so with varying specificity and affinity. For example, the alpha/v/beta/5 receptor on lung carcinoma cells binds VN alone, while the platelet glycoprotein GPIIb/IIIa the major receptor for fibrinogen, interacts with von Willebrand factor, fibronectin and VN. The mechanism of receptor specificity remain controversial. One hypothesis suggests that amino acid sequences adjacent to the RGD site determine tertiary structure thereby imparting specificity while another hypothesis suggests additional specific binding sites. The objective of this proposal is to study the structure/function relationships of VN by the use of recombinant DNA techniques.
The specific aims are: (1) to define the essential amino acid sequences, in addition to RGD, necessary for recognition by VN receptors alpha/v/beta/5, alpha/v/beta/3, and GPIIb/IIIa; (2) to determine the role of VN in tumor cell motility; and (3) to identify the PAI-I binding domain in VN. Site-directed and deletion mutagenesis and Bal31 nuclease digestion will be used to generate altered proteins which will be tested for cell adhesion and PAI-1 binding. In addition, monoclonal antibodies and peptide fragments will be used to characterize the PAI-1 binding domain. Recombinant human VN and two mutants of the RGD sequence have bene successfully expressed and secreted in baby hamster kid cells. The isolated recombinant wild-type VN promotes cell adhesion similar to plasma- derived VN. This demonstrates a viable system to study the questions raised.
| Cherny, R C; Honan, M A; Thiagarajan, P (1993) Site-directed mutagenesis of the arginine-glycine-aspartic acid in vitronectin abolishes cell adhesion. J Biol Chem 268:9725-9 |
