Adhesive properties of the β3 integrins: Comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells

Abstract

Glycoprotein IIb-IIIa (αIIbβ3) and the vitronectin receptor (αvβ3), two integrins that share the common β3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von 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-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP lib into M21-L cells, a variant of M21 cells (Cheresh, D. A., and R. C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional αv and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP lib were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/ GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking αvβ3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing αvβ3) 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-IIIa 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 IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor. Our results therefore demonstrate (a) that while GP IIb-IIIa 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 IIb-IIIa expressed in nucleated cells has similar adhesive properties as does GP IIb-IIIa in resting platelets but is not activated by platelet stimuli.