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
alpha3beta1 integrin to bind to models of the alpha1(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
alpha3beta1 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
beta1 integrin subunit binding to
collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3
integrin subunit
monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of
melanoma cells with single-stranded or triple-helical
peptide models of alpha1(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, alpha1(IV)531-543, can induce
alpha3beta1 integrin-mediated signal transduction in
melanoma cells and (ii)
ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several
alpha3beta1 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.