Regulation of Integrin Function by CD47 Ligands

Abstract

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...

Cholesterol-independent Interactions with CD47 Enhance αvβ3 Avidity

“…Furthermore, a recent report has shown that treatment with a similar peptide can increase ␣ v ␤ 3 -mediated adhesion (25). However, we saw no effect of 4N1K on the binding of LIBS antibodies to OV-10 cells expressing either wild-type or IgV-GPI, either alone or with GRGDSP treatment (not shown), indicating that the peptide did not influence RGD-induced activation.…”

“…We therefore tested the ability of 4N1K to enhance ␣ v ␤ 3 avidity by measuring OV-10 cell binding to AP3-coated plates, at coating concentrations that supported only nonspecific attachment by untreated cells. Although 4N1K has been reported to have CD47-independent effects on integrin functions in some situations (25,26), no increased binding with peptide treatment was observed for CD47-deficient cells (Fig. 3B).…”

“…Importantly, ligation of CD47 by TSP-derived agonist peptides such as 4N1K and 7N3 (VVM motif peptides) seems to promote both pathways of integrin activation. We and others (4,25,34) have reported that these agonist peptides act via a G i -dependent signaling pathway to augment integrin functions. In addition, there have been reports that the agonist peptides can augment integrin function even when G i is inactivated by pertussis toxin, suggesting that there may be G protein-independent FIG.…”

Regulation of Integrin Activity by MIA

“…For both ␣4 and ␣5, it has been shown that only the processed form is present on the membrane surface of cells (16,22,23). The antibodies used in our experiment were directed against the C termini of ␣4 and ␣5 integrins (16,18,24). In RIPA lysates of human melanocytes and Mel Im cells, the processed C-terminal fragment of ␣4 integrin was detected (Fig.…”

Heparan Sulfate Modification of the Transmembrane Receptor CD47 Is Necessary for Inhibition of T Cell Receptor Signaling by Thrombospondin-1

“…Engaging each of these receptors elicits specific signals in T cells (25,27). Somatic mutants of the Jurkat T cell line lacking ␤1 integrins or CD47 have been useful to define signaling pathways mediated by these thrombospondin receptors (27,30). However, further defining how TSP1 and TSP2 regulate T cell function is limited by not knowing the identity of the HSPG receptor.…”