Cholesterol-independent Interactions with CD47 Enhance α_vβ₃ Avidity^*

Abstract

Expression in OV10 cells of either wild-type CD47 or its extracellular IgV domain linked to a glycosylphosphatidylinositol anchor-(IgV-GPI) enhanced ligand-induced α_vβ₃ activation as detected by the binding of LIBS1 and LIBS6 mAbs. The amplitude of LIBS binding was greater with both CD47 and IgV-GPI expression, indicating an increase in the population of “activable” integrin molecules. Expression of either CD47 species also increased α_vβ₃-mediated adhesion to vitronectin, and to surfaces coated with the anti-β₃ antibody AP3, because of enhanced clustering of α_vβ₃ as confirmed by chemical cross-linking. Cholesterol depletion with methyl-β-cyclodextrin did not prevent the increase in anti-LIBS binding, but reduced cell adhesion to vitronectin and AP3. However, cells expressing CD47 were partially insulated against this disruption, and IgV-GPI was even more effective. Both CD47 and IgV-GPI were found in cholesterol-rich rafts prepared in the absence of detergent, but only CD47 could recruit α_vβ₃ and its associated signaling molecules to these domains. Thus CD47-α_vβ₃ complexes in cholesterol-rich raft domains appear to engage in G_i-dependent signaling whereas CD47-α_vβ₃ interactions that lead to integrin clustering are also detergent resistant, but are insensitive to cholesterol depletion and do not require the transmembrane region of CD47.