Ligation of Integrin α₅β₁ Is Required for Internalization of Vitronectin by Integrin α_vβ₃*

Abstract

Remodeling of the matrix by tumor cells is necessary for tumor invasion. We have shown previously that malignant astrocytomas, in contrast to normal astrocytes, synthesize vitronectin and express integrins α_vβ₃ and α_vβ₅. The activity states of these two integrins are differentially controlled. Thus, we investigated the regulation of the activity of integrins α_vβ₃ and α_vβ₅ with regard to their role in vitronectin internalization in U-251MG astrocytoma cell monolayers adherent to fibronectin, collagen, or laminin in serum-free conditions. Binding of [¹²⁵I]vitronectin occurred in a specific, saturable manner that was partially inhibitable by monoclonal antibodies (mAbs) specific for integrins α_vβ₃ or α_vβ₅. Specific, lysosomally-mediated degradation of [¹²⁵I]vitronectin was detectable at 1 h and increased over the 24-h assay period. The cell substrate affected the rate of turnover of [¹²⁵I]vitronectin, which was 3.0 ng/min for cells plated on fibronectin but 0.35 ng/min for cells plated on collagen. Furthermore, although mAbs specific for either integrin α_vβ₃ or α_vβ₅ inhibited degradation (30%; combined effect 70%) of [¹²⁵I]vitronectin by cells plated on fibronectin, only mAb anti-α_vβ₅ inhibited degradation (70-90%) by cells plated on collagen or laminin. To determine the requirement for integrin α₅β₁ ligation in order for integrin α_vβ₃ to internalize its ligand, cells were plated on mAbs anti-integrin α₅ or anti-integrin α₃. When plated on mAb anti-α₅, mAbs anti-α_vβ₃ and anti-α_vβ₅ both inhibited degradation. However, when plated on mAb anti-α₃, mAb anti-α_vβ₃ had no effect whereas mAb anti-α_vβ₅ inhibited degradation. These data indicate that a signal from integrin α₅β₁ is necessary for integrin α_vβ₃ to internalize vitronectin, whereas integrin α_vβ₅ constitutively internalizes vitronectin.