Membrane Raft Association of CD47 Is Necessary for Actin Polymerization and Protein Kinase C θ Translocation in Its Synergistic Activation of T Cells

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CD47 is a surface receptor that induces either coactivation or apoptosis in lymphocytes, depending on the ligand(s) bound. Interestingly, the apoptotic pathway is independent of caspase activation and cytochrome c release and is accompanied by early mitochondrial dysfunction with suppression of mitochondrial membrane potential (⌬⌿m). Using CD47 as bait in a yeast twohybrid system, we identified the Bcl-2 homology 3 (BH3)-only protein 19 kDa interacting protein-3 (BNIP3), a pro-apoptotic member of the Bcl-2 family, as a novel partner. Interaction between CD47 and the BH3-only protein was confirmed by immunoprecipitation analysis, and CD47-induced apoptosis was inhibited by attenuating BNIP3 expression with antisense oligonucleotides. Finally, we showed that the C-terminal domain of thrombospondin-1 (TSP-1), but not signal-regulatory protein (SIRP␣1), is the ligand for CD47 involved in inducing cell death. Immunofluorescence analysis of CD47 and BNIP3 revealed a partial colocalization of both molecules under basal conditions. After T cell stimulation via CD47, BNIP3 translocates to the mitochondria to induce apoptosis. These results show that the BH3-dependent apoptotic pathways, previously shown to be activated by intracellular pro-apoptotic events, can also be turned on by surface receptors. This new pathway results in a fast induction of cell death resembling necrosis, which is likely to play an important role in lymphocyte regulation at inflammatory sites and/or in the vicinity of thrombosis.

Section: Methodsmentioning

confidence: 99%

CD47 and the 19 kDa Interacting Protein-3 (BNIP3) in T Cell Apoptosis

Lamy

Ticchioni

Rouquette-Jazdanian

et al. 2003

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“…As previously demonstrated for ␤ 3 integrins and ␣ 2 ␤ 1 , CD47 is physically associated with ␣ 4 ␤ 1 in cells, where it regulates function of this ␤ 1 integrin. Both CD47 (40,41) and ␣ 4 ␤ 1 integrin (42) associate with lipid rafts in Jurkat cells, suggesting that their interaction may involve these membrane microdomains. Notably, ␣ v ␤ 3 activity is increased when CD47 translocates out of raft domains (43).…”

Section: A Cd47-binding Peptide Stimulatesmentioning

confidence: 99%

Regulation of Integrin Function by CD47 Ligands

Barazi

Cashel

et al. 2002

CD47 (integrin-associated protein) is an integral membrane protein that is required for granulocyte and T cell recruitment to sites of infection (1, 2), and its absence on red blood cells leads to their rapid macrophage-mediated clearance (3). CD47 may also function as a costimulator to regulate T cell activation, survival, and Th1 versus Th2 differentiation (4, 5). Endogenous ligands for the extracellular domain of CD47 include the secreted protein thrombospondin-1 (TSP1) 1 and potentially other members of the thrombospondin family, several integrins (6 -9), and some members of the signal-regulatory protein family (3, 10 -13). Engagement of CD47 by soluble ligands or signal-regulatory protein counter receptors modulates several cell signaling pathways, including activation of a heterotrimeric G protein (14).Although some signal transduction through CD47 is integrin-independent (4, 15, 16), association of CD47 with certain integrins was found to modulate their function to mediate cell adhesion or motility (6,8,9,17). In addition to its known functional and physical interactions with ␣ v ␤ 3 , ␣ IIb ␤ 3 , and ␣ 2 ␤ 1 integrins, several publications have suggested an association of CD47 with ␣ 4 ␤ 1 integrin. CD47 and ␣ 4 ␤ 1 were colocalized on microvilli of K562 erythroleukemia cells (18), and CD47-dependent arrest of T cells on inflammatory endothelium could be blocked by antibodies that prevent ␣ 4 ␤ 1 integrin binding to VCAM-1 (2). In the latter study, ligation of CD47 by TSP1 or signal-regulatory protein 1␣ was inferred to activate ␣ 4 ␤ 1 integrin on T cells, although this was not demonstrated either functionally or biochemically.We recently found that CD47 expression is required for stimulation of T cell motility and expression of MMP-2 stimulated by ␣ 4 ␤ 1 integrin ligands (19). An antibody to CD47 also blocked the motility response to an ␣ 4 ␤ 1 integrin ligand (19). Therefore, at least a functional interaction occurs between CD47 and ␣ 4 ␤ 1 integrin.We identified a binding site for ␣ 4 ␤ 1 integrin in the Nterminal domains of TSP1 and TSP2 (19), whereas two binding sites for CD47 have been localized to the C-terminal domain of TSP1 (for review, see Ref. 20). Thus, TSP1 could potentially regulate its own interaction with ␣ 4 ␤ 1 integrin through simultaneous interactions with CD47 and ␣ 4 ␤ 1 integrin on a T cell, analogous to the ability of soluble TSP1 to stimulate ␣ v ␤ 3 integrin-dependent melanoma cell adhesion to immobilized TSP1 (21).To define a molecular basis for functional cross-talk between these two receptors, we have examined the effect of CD47 ligands on the function of ␣ 4 ␤ 1 integrin in T cells and melanoma cells. We confirmed that ligation of CD47 can activate ␣ 4 ␤ 1 integrin function but unexpectedly found the mechanism of this activity to be CD47 ligand-dependent. We further show that CD47 ligation differentially activates and inactivates ␣ v ␤ 3 and ␣ 4 ␤ 1 in melanoma cells and ␣ 4 ␤ 1 and ␣ 5 ␤ 1 in Jurkat cells. Unexpectedly, but consistent with a recent report demonstrating C...

“…CD47 was expressed in these cells in its wild-type form and as a fusion protein containing the IgV domain linked to a glycophosphatidylinositol anchor (IgV-GPI) (13). As compared with wild-type CD47, the IgV-GPI species has been shown to be deficient in downstream T cell signaling induced by CD47 ligation (22) (10). On the other hand, as discussed above, IgV-GPI supported vitronectin-coated bead binding when co-expressed with ␣v␤3, and so may serve as an indicator of signaling-independent effects of CD47 (13).…”

Section: Binding Of Libs Antibodies To ␣ V ␤ 3 Is Enhanced By Cd47mentioning

confidence: 99%

“…CD47-mediated signaling also occurs in Jurkat T cells, where treatment with certain anti-CD47 antibodies promotes apoptosis via a G i -mediated pathway involving cyclic AMP regulation of cAMP-dependent kinase (11). Ligation of CD47 with other antibodies has been shown to have a synergistic effect on T cell activation that required the MMS domain of CD47 for both the T cell response and for raft localization (10). Thus raft localization may play a role in organizing CD47 signaling complexes in a number of systems.…”

mentioning

confidence: 99%

“…The ␣ v ␤ 3 -CD47-G i complex has also been found in Triton X-100-resistant membrane domains, or rafts, which are thought to act as organizing platforms for diverse signaling pathways, including those involving growth factors, G proteincoupled receptors, and T cell activation (9,10). Depletion of membrane cholesterol was shown to destabilize the ␣ v ␤ 3 -CD47-G i complex in C32 melanoma and ovarian carcinoma cells, suggesting that intramembrane contacts controlling its localization to rafts may promote or even be required for complex assembly and G i -coupled signaling (9).…”

mentioning

confidence: 99%

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Cholesterol-independent Interactions with CD47 Enhance αvβ3 Avidity

McDonald

Zheleznyak

Frazier

2004

Expression in OV10 cells of either wild-type CD47 or its extracellular IgV domain linked to a glycosylphosphatidylinositol anchor-(IgV-GPI) enhanced ligand-induced ␣ v ␤ 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 ␣ v ␤ 3 -mediated adhesion to vitronectin, and to surfaces coated with the anti-␤ 3 antibody AP3, because of enhanced clustering of ␣ v ␤ 3 as confirmed by chemical cross-linking. Cholesterol depletion with methyl-␤-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 ␣ v ␤ 3 and its associated signaling molecules to these domains. Thus CD47-␣ v ␤ 3 complexes in cholesterol-rich raft domains appear to engage in G i -dependent signaling whereas CD47-␣ v ␤ 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.