Effect of Ligand Conformation on Melanoma Cell α3β1Integrin-Mediated Signal Transduction Events: Implications for a Collagen Structural Modulation Mechanism of Tumor Cell Invasion

Abstract

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the α₃β₁ integrin to bind to models of the α1(IV)531−543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem.269, 30939−30945; Miles, A. J., et al. (1995) J. Biol. Chem.270, 29047−29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by α₃β₁ integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125^FAK, consistent with prior studies correlating β₁ integrin subunit binding to collagen and p125^FAK Tyr phosphorylation. Cross-linking of an anti-α₃ integrin subunit monoclonal antibody also induced p125^FAK Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of α1(IV)531−543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125^FAK. Induction of p125^FAK Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125^FAK phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, α1(IV)531−543, can induce α₃β₁ integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several α₃β₁ integrin-mediated signal transduction events. The effects of ligand conformation suggest that a “collagen structural modulation” mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.