IMMUNOMORPHOLOGICAL CHARACTERIZATION AND EFFECTS OF 12-(S)-HETE ON A DYNAMIC INTRACELLULAR POOL OF THE ALPHA(IIB)BETA(3)-INTEGRIN IN MELANOMA-CELLS

In metastatic B16a murine melanoma cells, alpha(IIb)beta(3) integrin w as shown to be one of the key adhesion molecules responsible for matri x adhesion and spreading. Upon stimulation, alpha(IIb)beta(3) can be u pregulated at the cell surface due to translocation of the receptor to the plasma membrane from an intracenular pool. Here we have character ized this integrin pool as a tubulovesicular structure (TVS) correspon ding to endosomes. TVS was found to be associated temporarily with mic rotubules and intermediate filaments especially after protein kinase C (PKC) stimulation with a lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. After PKC stimulat ion, the predominantly vesicular TVS became elongated and alpha(IIb)be ta(3) appeared at the apical plasma membrane and microvilli. Disruptio n of either the microtubules or intermediate filaments prevented the 1 2-(S)-HETE effect both on vesicular to tubular transition of TVS as we ll as on surface expression of this integrin. The connection with the Golgi system of the integrin-containing TVS was proved by a Golgi-inhi bitor (brefeldin A) pretreatment, which prevented the PKC-stimulation- induced TVS elongation and subsequent receptor-upregulation at the cel l surface. After a soluble ligand binding (mAb to the alpha(IIb)beta(3 ) complex) the surface receptor endocytosed back to the TVS indicating the presence of a dynamic, cytoskeleton associated integrin pool in m elanoma cells.