Spreading and Focal Contact Formation of Human Melanoma Cells in Response to the Stimulation of Both Melanoma-associated Proteoglycan (NG2) and α4β1 Integrin

In this study, we evaluated the potential role for a specific melanoma-associated chondroitin sulfate proteoglycan core protein, termed NG2, to collaborate with α4β1 integrin in focal contact formation in human melanoma cells. Although melanoma cells adhered to substrata coated with either the α4β1 integrin binding fibronectin synthetic peptide CS1-OVA or anti-NG2 mAbs, no spreading or focal contact formation was observed on either substratum. However, melanoma cells spread and formed focal contacts on “chimeric substrata” coated with CS1-OVA and the anti-NG2 mAb, 9.2.27, indicating that engaging both adhesion receptors changes the adhesion phenotype of melanoma cells by reorganizing the cytoskeleton. The collaboration between the two receptors is specific to fibronectin, since cells adherent on substrata coated with low concentrations of either laminin and 9.2.27 or type IV collagen and 9.2.27 failed to spread, while cells adherent on low concentrations of fibronectin and 9.2.27 exhibited a fully spread morphology. Two selective tyrosine kinase inhibitors, genistein and herbimycin A, totally inhibited cell spreading on the substrata coated with CS1-OVA and 9.2.27, indicating that tyrosine kinase(s) is important for cell spreading and focal contact formation. When cells were cultured on substrata coated with CS1-OVA and 9.2.27, two proteins (Mr 130,000 and 120,000) were tyrosine phosphorylated in a genistein- and herbimycin A-sensitive fashion. These proteins were not immunologically related to ppl25FAK or α4β1 integrin. Importantly, when melanoma cells were cultured on substrata coated with CS1 and then stimulated with 9.2.27-conjugated microsphere beads, formation of focal contacts and stress fibers was also observed, indicating that NG2 can collaborate with α4β1 integrin when each receptor is engaged on distinct and separate substrata. These results demonstrate that NG2 acts as a coreceptor for spreading and focal contact formation in association with α4β1 integrin in melanoma cells and suggest a model in which the NG2 core protein communicates to α4β1 integrin by an inside-out signaling mechanism.