SPREADING AND FOCAL CONTACT FORMATION OF HUMAN-MELANOMA CELLS IN RESPONSE TO THE STIMULATION OF BOTH MELANOMA-ASSOCIATED PROTEOGLYCAN (NG2)AND ALPHA-4-BETA-1 INTEGRIN

In this study, we evaluated the potential role for a specific melanoma -associated chondroitin sulfate proteoglycan core protein, termed NG2, to collaborate with alpha 4 beta 1 integrin in focal contact formatio n in human melanoma cells. Although melanoma cells adhered to substrat a coated with either the alpha 4 beta 1 integrin binding fibronectin s ynthetic peptide CS1-OVA or anti-NG2 mAbs, no spreading or focal conta ct formation was observed on either substratum. However, melanoma cell s spread and formed focal contacts on ''chimeric substrata'' coated wi th CS1-OVA and the anti-NG2 mAb, 9.2.27, indicating that engaging both adhesion receptors changes the adhesion phenotype of melanoma cells b y reorganizing the cytoskeleton. The collaboration between the two rec eptors is specific to fibronectin, since cells adherent on substrata c oated 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 conc entrations of fibronectin and 9.2.27 exhibited a fully spread morpholo gy. 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 ce ll spreading and focal contact formation. When cells were cultured on substrata coated with CS1-OVA and 9.2.27, two proteins (M(r) 130,000 a nd 120,000) were tyrosine phosphorylated in a genistein- and herbimyci n A-sensitive fashion, These proteins were not immunologically related to pp125(FAK) or alpha 4 beta 1 integrin. Importantly, when melanoma cells were cultured on substrata coated with CS1 and then stimulated w ith 9.2.27-conjugated microsphere beads, formation of focal contacts a nd stress fibers was also observed, indicating that NG2 can collaborat e with alpha 4 beta 1 integrin when each receptor is engaged on distin ct and separate substrata, These results demonstrate that NG2 acts as a coreceptor for spreading and focal contact formation in association with alpha 4 beta 1 integrin in melanoma cells and suggest a model in which the NG2 core protein communicates to alpha 4 beta 1 integrin by an inside-out signaling mechanism.