The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18

Gupta, Vineet; Gylling, Annette; Alonso, José L.; Sugimori, Takashi; Ianakiev, Petre; Xiong, Jiang-Ping; Arnaout, M. Amin

doi:10.1182/blood-2005-11-056689

Cited by 53 publications

(57 citation statements)

References 50 publications

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“…It has been reported that replacing the ␤2 CD loop sequence with the homologous ␤3 sequence or inserting a N-glycan-binding site in the CD loop in ␣M␤2 integrin induced robust ligand binding (27). However, the fact that the deletion of the CD loop of the ␤T domain failed to activate ␣IIb␤3 in our experiment strongly argues against the deadbolt theory, in which an endogenous ␤A/␤T interface interaction plays a critical role in maintaining integrin in its low affinity state.…”

Section: Discussioncontrasting

confidence: 55%

Structural Requirements for Activation in αIIbβ3 Integrin

Kamata

Handa

Ito

et al. 2010

Journal of Biological Chemistry

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Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant ␣IIb␤3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant ␣IIb␤3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the ␤-head/␤-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the ␣-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the ␣-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.Integrin-mediated bidirectional signaling is closely associated with the structural rearrangement of integrin itself. During inside-out signaling, talin has been shown to bind to the ␤ cytoplasmic tail and to disrupt the endogenous interaction between the ␣ and ␤ cytoplasmic tails (1, 2). The dissociation of the two tails induces a structural rearrangement of the extracellular domains increasing the affinity to the ligand. During outside-in signaling, ligand binding in turn induces the structural rearrangement of the extracellular domains. This structural change propagates through the plasma membrane to separate the cytoplasmic tails, providing binding sites for numerous cytoplasmic proteins (3). Thus, the two structures flanking the plasma membrane affect each other. This structural rearrangement can be detected using a group of monoclonal antibodies (mAbs) that bind preferentially to the ligand-bound form (4). These anti-LIBS 2 (ligand-induced binding site) mAbs have been used not only to investigate the activation status of a specific integrin but also to activate it (5). However, without information on the actual three-dimensional structure, it is impossible to determine the specific conformation recognized by each anti-LIBS mAb.The first observation of the actual three-dimensional structure of integrin was made using conventional electron ...

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Section: Discussioncontrasting

confidence: 55%

Structural Requirements for Activation in αIIbβ3 Integrin

Kamata

Handa

Ito

et al. 2010

Journal of Biological Chemistry

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“…Therefore, we conclude that the ␤-tail domain CD loop does not regulate ligand binding and does not act as a deadbolt. Recently, it was reported that exchanging D658GMD in the ␤-tail domain CD loop of the ␤2 subunit with D672SSG of the ␤3 subunit or with N658GTD, which introduces a glycan wedge sequence, activated the ␣M␤2 integrin (43). A crystal structure of ␣M␤2 is not available.…”

Section: Discussionmentioning

confidence: 99%

Tests of the Extension and Deadbolt Models of Integrin Activation

Zhu

Boylan

Luo

et al. 2007

Journal of Biological Chemistry

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Despite extensive evidence that integrin conformational changes between bent and extended conformations regulate affinity for ligands, an alternative hypothesis has been proposed in which a "deadbolt" can regulate affinity for ligand in the absence of extension. Here, we tested both the deadbolt and the extension models. According to the deadbolt model, a hairpin loop in the ␤3 tail domain could act as a deadbolt to restrain the displacement of the ␤3 I domain ␤6-␣7 loop and maintain integrin in the low affinity state. We found that mutating or deleting the ␤3 tail domain loop has no effect on ligand binding by either ␣IIb␤3 or ␣V␤3 integrins. In contrast, we found that mutations that lock integrins in the bent conformation with disulfide bonds resist inside-out activation induced by cytoplasmic domain mutation. Furthermore, we demonstrated that extension is required for accessibility to fibronectin but not smaller fragments. The data demonstrate that integrin extension is required for ligand binding during integrin inside-out signaling and that the deadbolt does not regulate integrin activation.Integrins are cell adhesion molecules that transmit bidirectional signals across the plasma membrane and regulate many biological functions, including cell differentiation, cell migration, and wound healing (1, 2). A bent conformation observed by x-ray crystallography (3) and electron microscopy (4, 5) represents the physiological low affinity state. Integrin activation is regulated by binding of intracellular proteins such as talin to the integrin ␤-subunit cytoplasmic tail (6), leading to separation between the ␣-and ␤-subunits at their transmembrane and cytoplasmic domains (7-9) and propagation of conformational changes from the transmembrane domains to the ligand binding headpiece, which increases integrin affinity for ligand (10,11). These long range conformational changes involve integrin extension. Conversely, ligand binding also induces integrin extension, which leads to the separation of the ␣-and ␤-subunit legs and the transmembrane and cytoplasmic regions (11).Recent co-crystal structures of integrin ␣IIb␤3 headpiece bound to ligands revealed the molecular basis for the large conformational changes that accompany ligand binding (12). The conformational changes in the ligand binding site that increase affinity for ligand are allosterically linked by a crankshaft-like ␣7-helix displacement in the ␤3 I domain to a 60°change in the angle between the ␤3 I and hybrid domains and to a 70 Å increase in separation between the ␣-and ␤-subunits at their knees. Since the hybrid domain and connecting ␤-leg domains are central in the interface between the integrin headpiece and legs in the bent conformation, integrin extension enables hybrid domain swingout. The crystal structures are in excellent agreement with studies using electron microscopy (4, 5, 13, 14), NMR (15), small angle x-ray scattering (16), and mutational studies and mapping of the epitopes of allosteric activating and inhibiting antibodies (17)(18)(19)(20), ...

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“…It has been postulated that the CDloop acts as a deadbolt, preventing integrin activation by locking βA in an inactive state. Indeed, a number of antibodies that activate β1 integrins bind to the βTD or the βA interface, and the βTD has been shown to regulate ligand binding (27,29). We propose that asTF induces the removal of the CD-loop deadbolt.…”

Section: Discussionmentioning

confidence: 99%

“…We hypothesize that asTF induces a conformational change in β1 integrins that render them prone to activation, as we used a β1 integrin-blocking antibody that is reactive with the membrane-proximal β-tail domain (βTD) of the β1 integrin subunit, and the 579-799 integrin peptide features this domain. The βTD contains a CD-loop that contacts the ligand-binding integrin βA domain and the hybrid domain, and this contact is lost upon integrin activation (27,28). It has been postulated that the CDloop acts as a deadbolt, preventing integrin activation by locking βA in an inactive state.…”

Section: Discussionmentioning

confidence: 99%

Alternatively spliced tissue factor promotes breast cancer growth in a β1 integrin-dependent manner

Kocatürk¹,

Berg²,

Tieken³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

126

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Full-length tissue factor (flTF), the coagulation initiator, is overexpressed in breast cancer (BrCa), but associations between flTF expression and clinical outcome remain controversial. It is currently not known whether the soluble alternatively spliced TF form (asTF) is expressed in BrCa or impacts BrCa progression. We are unique in reporting that asTF, but not flTF, strongly associates with both tumor size and grade, and induces BrCa cell proliferation by binding to β1 integrins. asTF promotes oncogenic gene expression, anchorage-independent growth, and strongly up-regulates tumor expansion in a luminal BrCa model. In basal BrCa cells that constitutively express both TF isoforms, asTF blockade reduces tumor growth and proliferation in vivo. We propose that asTF plays a major role in BrCa progression acting as an autocrine factor that promotes tumor progression. Targeting asTF may comprise a previously unexplored therapeutic strategy in BrCa that stems tumor growth, yet does not impair normal hemostasis.regulated pre-mRNA processing | outside-in signaling I n breast cancer (BrCa), proteins that modulate splicing events such as ASF/SF2 and SR(Serine/Arginine-rich)p55, are frequently up-regulated and contribute to cell transformation (1, 2). BrCa cells exhibit specific alternative splicing signatures that were proposed as potential prognostic factors in BrCa (3). Alternative splicing of proteins, such as spleen tyrosine kinase (Syk), p53, phosphatase and tensin homolog (PTEN), chemokine (C-X-C motif) receptor 3 (CXCR3), and ras-related C3 botulinum toxin substrate 1 (Rac1) impacts BrCa cell behavior and therefore, disease progression (4-8).Full-length tissue factor (flTF) is the initiator of blood coagulation (9). Following vascular damage, flTF binds its ligand FVII(a), which triggers clot formation. Aside from subendothelial tissues, flTF is also abundant on cancer cells (9) and fuels tumor progression by modulating integrin α3β1 function, cell migration (10), and FVIIa-dependent protease activated receptor (PAR)2 activation, but flTF-β1 integrin complexation enhances PAR2 activation (11). flTF-dependent PAR2 activation results in the production of VEGF, CXCL1, and IL-8, thus promoting the angiogenic switch and consequently, tumor growth in vivo (10, 11).Alternative splicing of TF pre-mRNA results in the deletion of exon 5 and thus a frameshift in exon 6, yielding a transmembrane domain-lacking isoform that can be secreted (12). Human and murine alternatively spliced TF (asTF) contain novel C termini with poor homology to one another or any other protein, and the murine asTF C terminus is longer than that of human asTF (12,13). High expression of asTF in tumor cell lines suggests a role in tumor progression (10,14). Subcutaneous growth of pancreatic cancer cells overexpressing asTF results in larger and more vascularized tumors (15). We recently discovered that asTF induces angiogenesis, independent of PAR2 activation, by acting as an integrin ligand (16). Thus, flTF and asTF facilitate cellular signaling via ...

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The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18

Cited by 53 publications

References 50 publications

Structural Requirements for Activation in αIIbβ3 Integrin

Structural Requirements for Activation in αIIbβ3 Integrin

Tests of the Extension and Deadbolt Models of Integrin Activation

Alternatively spliced tissue factor promotes breast cancer growth in a β1 integrin-dependent manner

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