Ligation of integrin alpha5beta1 is required for internalization of vitronectin by integrin alphavbeta3

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 alphavbeta3 and alphavbeta5. The activity states of these two integrins are differentially controlled. Thus, we investigated the regulation of the activity of integrins alphavbeta3 and alphavbeta5 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 alphavbeta3 or alphavbeta5. 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 alphavbeta3 or alphavbeta5 inhibited degradation (30%; combined effect 70%) of [125I]vitronectin by cells plated on fibronectin, only mAb anti-alphavbeta5 inhibited degradation (70-90%) by cells plated on collagen or laminin. To determine the requirement for integrin alpha5beta1 ligation in order for integrin alphavbeta3 to internalize its ligand, cells were plated on mAbs anti-integrin alpha5 or anti-integrin alpha3. When plated on mAb anti-alpha5, mAbs anti-alphavbeta3 and anti-alphavbeta5 both inhibited degradation. However, when plated on mAb anti-alpha3, mAb anti-alphavbeta3 had no effect whereas mAb anti-alphavbeta5 inhibited degradation. These data indicate that a signal from integrin alpha5beta1 is necessary for integrin alphavbeta3 to internalize vitronectin, whereas integrin alphavbeta5 constitutively internalizes vitronectin.