Hierarchy of ADAM12 binding to integrins in tumor cells

Abstract ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that α9β1 integrin is the main receptor for ADAM12. However, when α9β1 integrin is not expressed–as in many carcinoma cells–other members of the β1 integrin family can replace its ligand binding activity. In attachment assays, the recombinant disintegrin domain of ADAM12 only supported α9 integrin-dependent tumor cell attachment, whereas full-length ADAM12 supported attachment via α9 integrin and other integrin receptors. Cells that attached to full-length ADAM12 in an α9 integrin-dependent manner also attached to ADAM12 in which the putative α9β1 integrin-binding motif in the disintegrin domain had been mutated. This attachment was mediated through use of an alternate β1 integrin. We also found that cell spreading in response to ADAM12 is dependent on the apparent level of integrin activation. Binding of cells to ADAM12 via the α9β1 integrin was Mn 2+ -independent and resulted in attachment of cells with a rounded morphology; attachment of cells with a spread morphology required further activation of the α9β1 integrin. We demonstrated that phosphoinositide-3-kinase appears to be central in regulating α9β1 integrin cell spreading activity in response to ADAM12.