Adhesive Properties of the/ 3 Integrins: Comparison of GP IIb-IIIa and the Vitronectin Receptor Individually Expressed in Human Melanoma Cells

Glycoprotein llb-IRa (ctnbB3) and the vitronectin receptor (ct~B3), two integrins that share the common #3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, yon Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-iRa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP Hb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and yon Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP lab into M21-L cells, a variant of M21 cells (Cheresh, D. A., and R. C. Spiro. 1987. J. Biol. Chem. 262:1770317711), which lack the expression of functional ctv and are therefore unable to attach to vitronectin, fibrinogen, and yon Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP lib-IRa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/ GP n'b cells. Comparative functional studies demonstrated that GP llb-IIIa expressed in M21-L/GPIlb cells as well as native GP Ilb-tlla constitutively expressed in HEL-5J20 cells (an HEL variant lacking ctv~3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von WiUebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing c~v/33) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-iRa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP Ilb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor. Our results therefore demonstrate (a) that while GP IIb-I]Ia in its inactive, resting form is capable of mediating adhesion of platelets to immobilized fibrinogen, it does not to other RGD-containing adhesive proteins such as von Willebrand factor and vitronectin, and (b) that GP Ilb-iRa expressed in nucleated cells has similar adhesive properties as does GP llbIRa in resting platelets but is not activated by platelet