Cooperation between Thrombospondin-1 Type 1 Repeat Peptides and αvβ3 Integrin Ligands to Promote Melanoma Cell Spreading and Focal Adhesion Kinase Phosphorylation

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In addition to the three known ␤ 1 integrin recognition sites in the N-module of thrombospondin-1 (TSP1), we found that ␤ 1 integrins mediate cell adhesion to the type 1 and type 2 repeats. The type 1 repeats of TSP1 differ from typical integrin ligands in that recognition is pan-␤ 1 -specific. Adhesion of cells that express one dominant ␤ 1 integrin on immobilized type 1 repeats is specifically inhibited by antagonists of that integrin, whereas adhesion of cells that express several ␤ 1 integrins is partially inhibited by each ␣-subunit-specific antagonist and completely inhibited by combining the antagonists. ␤ 1 integrins recognize both the second and third type 1 repeats, and each type 1 repeat shows pan-␤ 1 specificity and divalent cation dependence for promoting cell adhesion. Adhesion to the type 2 repeats is less sensitive to ␣-subunit antagonists, but a ␤ 1 blocking antibody and two disintegrins inhibit adhesion to immobilized type 2 repeats. ␤ 1 integrin expression is necessary for cell adhesion to the type 1 or type 2 repeats, and ␤ 1 integrins bind in a divalent cation-dependent manner to a type 1 repeat affinity column. The widely used TSP1 function blocking antibody A4.1 binds to a site in the third type 2 repeat. A4.1 proximally inhibits ␤ 1 integrin-dependent adhesion to the type 2 repeats and indirectly inhibits integrin-dependent adhesion mediated by the TSP1 type 1 repeats. Although antibody A4

Section: Discussionsupporting

confidence: 82%

Identification of Novel β1 Integrin Binding Sites in the Type 1 and Type 2 Repeats of Thrombospondin-1

Calzada

Annis

Zeng

et al. 2004

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“…An additional anti-␤ 3 mAb AP1 was purchased from GTI Inc. A functionally blocking mAb to ␤ 1 integrin, mAb 13, was kindly provided by Dr. Kenneth Yamada. Purified human thrombospondin-1 (TS1) and anti-TS1 polyclonal antibody R1 were kindly provided by Dr. Jack Lawler (31). Rabbit polyclonal antibody against human CD11b R7928A was raised by immunizing rabbit with a peptide (DMMSEGGPPGAEPQ) corresponding to the C terminus of CD11b.…”

Section: Methodsmentioning

confidence: 99%

The Role of CD47 in Neutrophil Transmigration

Liu

Merlin

Burst

et al. 2001

175

CD47, a cell surface glycoprotein, plays an important role in modulating neutrophil (PMN) migration across endothelial and epithelial monolayers. Here we show that anti-CD47 monoclonal antibodies (mAbs) delay PMN migration across collagen-coated filters or T84 epithelial monolayers toward the chemoattractant formylmethionylleucylphenylalanine (fMLP). Despite delayed transmigration by anti-CD47 mAbs, the numbers of PMN migrating across in either condition were the same as in the presence of control non-inhibitory mAbs. Cell surface labeling and immunoprecipitation demonstrated upregulation of CD47 to the PMN cell surface with kinetics similar to those of the transmigration response. Subcellular fractionation studies revealed redistribution of CD47 from intracellular compartments that co-sediment with secondary granules to plasma membrane-containing fractions after fMLP stimulation. Experiments performed to investigate potential signaling pathways revealed that inhibition of tyrosine phosphorylation with genistein reversed the anti-CD47-mediated PMN migration delay, whereas inhibition of phosphatidylinositol 3-kinase only partially reversed anti-CD47 effects that correlated with a rapid increase in PMN cell surface CD47. Analysis of the contribution of epithelial-expressed CD47 to PMN transmigration revealed that PMN migration across CD47-deficient epithelial monolayers (CaCO2) was significantly increased after stable transfection with CD47. These results suggest that cell surface CD47 and downstream tyrosine phosphorylation signaling events regulate, in part, the rate of PMN migration during the inflammatory response.

“…Vitrogen type I collagen was obtained from Cohesion, Palo Alto, CA. The following synthetic peptides derived from TSP1 and their respective inactive controls were prepared as previously described: FIRVVMYEGKK (7N3, residues 1102-1112 of mature TSP1), RFYVVMWK (4N1-1, 1016 -1024), KRFYVVMWKK (4N1K, 4N1 flanked with 2 Lys residues), RFYGGMWK (4N1GG, inactive control for 4N1), and FIRGGMYEGKK (p604) and FIRVAIYEGKK (p605), both controls for 7N3 (21,27).…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

Regulation of Integrin Function by CD47 Ligands

Barazi

Cashel

et al. 2002

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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...