Cholesterol-independent interactions with CD47 enhance alphavbeta3 avidity

• Published in: The Journal of biological chemistry Vol. 279; no. 17; p. 17301
• Main Authors: McDonald, John F, Zheleznyak, Alex, Frazier, William A
• Format: Journal Article
• Language: English
• Published: United States 23-04-2004
• Subjects: Antigens, CD - metabolism; Carrier Proteins - metabolism; CD47 Antigen; Cell Adhesion; Cell Line, Tumor; Cholesterol - chemistry; Cholesterol - metabolism; Cross-Linking Reagents - pharmacology; Cyclodextrins - pharmacology; Detergents - pharmacology; Dose-Response Relationship, Drug; Flow Cytometry; Glycosylphosphatidylinositols - chemistry; GTP-Binding Protein alpha Subunits, Gi-Go - metabolism; Humans; Integrin alphaVbeta3 - metabolism; Integrins - chemistry; Ligands; Membrane Microdomains - chemistry; Oligopeptides - chemistry; Protein Binding; Protein Structure, Tertiary; Signal Transduction; Vitronectin - chemistry
• ISSN: 0021-9258

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.