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 alpha(v)beta(3) 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 alpha(v)beta(3)-mediated adhesion to vitronectin, and to surfaces coated with the anti-beta(3) antibody AP3, because of enhanced clustering of alpha(v)beta(3) as confirmed by chemical cross-linking. Cholesterol depletion with methyl-beta-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 alpha(v)beta(3) and its associated signaling molecules to these domains. Thus CD47-alpha(v)beta(3) complexes in cholesterol-rich raft domains appear to engage in G(i)-dependent signaling whereas CD47-alpha(v)beta(3) 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.
Highlights
Integrins form complexes with other transmembrane proteins that can modulate their functions
Binding of ligand-induced binding sites (LIBS) Antibodies to ␣v3 Is Enhanced by CD47 Expression—To determine if CD47 expression can have a direct influence on ␣v3 activation, binding of antibodies recognizing LIBS was measured for OV10 cells co-transfected with CD47 and 3 integrin
RGD-induced binding of LIBS-1 was enhanced with expression of either CD47 species (Fig. 1, A and C), with IgV domain linked to a glycosylphosphatidylinositol anchor- (IgV-GPI) producing the largest effect
Summary
Integrins form complexes with other transmembrane proteins that can modulate their functions. A previous study had shown that expression of the CD47 extracellular IgV domain in ovarian carcinoma cells was required for ␣v3-mediated binding of vitronectin-coated beads [13]. These data indicate that the physical association of only the IgV domain of CD47 can affect 3 integrin function. CD47 may influence 3 functions through interactions that involve different regions of the molecule While it seems that signaling-dependent actions of CD47 may require cholesterol-rich domains, the possible role of membrane domains in more direct effects on the conformation of its associated integrin is unknown. CD47 interactions with ␣v3 were found to promote both integrin activation induced by RGD peptide, and ␣v3 avidity in binding immobilized substrates by
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