Characterization of the Cross-Linking Site of Disintegrins Albolabrin, Bitistatin, Echistatin, and Eristostatin on Isolated Human Platelet Integrin GpIIb/IIIa

Calvete, Juan J.; McLane, Mary Ann; Stewart, G. J.; Niewiarowski, Stefan

doi:10.1006/bbrc.1994.1903

Cited by 24 publications

(15 citation statements)

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“…These become activated by a trans‐autophosphorylation mechanism similar to that established for receptor tyrosine kinases. Echistatin is likely to directly interact with integrins [3,35,36]. It might promote a conformational change of integrin molecules that could affect the interaction of their β‐cytoplasmic tails with the amino terminus domain of pp125 FAK causing the subsequent block of pp125 FAK activation.…”

Section: Discussionmentioning

confidence: 99%

Echistatin inhibits pp125^FAK autophosphorylation, paxillin phosphorylation and pp125^FAK–paxillin interaction in fibronectin‐adherent melanoma cells

Morte¹,

Squillacioti²,

Garbi³

et al. 2000

European Journal of Biochemistry

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Echistatin, a snake-venom RGD-containing protein, was previously shown to disrupt cell-matrix adhesion by a mechanism that involves the reduction of pp125 FAK tyrosine phosphorylation levels. The aim of this study was to establish the sequence of events downstream pp125 FAK dephosphorylation that could be responsible for echistatin-induced disassembly of actin cytoskeleton and focal adhesions in fibronectin-adherent B16-BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosphorylation and kinase activity of pp125 FAK . One hour of cell exposure to echistatin caused a 39% decrease of pp125 FAK Tyr397 phosphorylation and a 31% reduction of pp125 FAK autophosphorylation activity as measured by immunecomplex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125 FAK . Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both paxillin and pp125 FAK from focal adhesions. The reduction of paxillin phosphorylation may represent a critical step in the pathway by which disintegrins exert their biological activity, including the inhibition of experimental metastasis in vivo.Keywords: echistatin; B16-BL6 melanoma cells; pp125 FAK ; paxillin; focal adhesions.Echistatin belongs to a family of low molecular mass, Arg-GlyAsp (RGD)-containing proteins isolated from the venom of various snakes [1±4]. These proteins are termed disintegrins for their ability to bind with high affinity integrin receptors on platelets and cell surfaces. Inhibition of platelet aggregation and of cell adhesion has been the first biological activity reported for disintegrins [5]. The ability of some disintegrins to inhibit experimental metastasis in vivo has also been proved [6±10]. The exact molecular mechanism by which disintegrins act is still unclear; however, the finding that disintegrins affect integrin-dependent cell signaling provided some new insights into it [11±13].We previously demonstrated that echistatin, a small-size disintegrin, inhibits B16-BL6 murine melanoma cell adhesion to extracellular matrix (ECM) components and detaches fibronectin-adherent melanoma cells from the substratum [13,14]. Echistatin-induced disruption of cell±matrix interactions in fibronectin-adherent B16-BL6 cells appears to involve an early reduction of pp125 FAK phosphorylation that correlates with the disassembly of actin cytoskeleton and of focal adhesions. However, the functional relationship between these events has not been established.The 68-kDa protein paxillin, a component of focal adhesions, is a substrate of pp125 FAK or of a kinase activated by pp125 FAK [15]. The integrin-dependent phosphorylation of pp125 FAK and paxillin has been observed in many cell types [16]. Elevated content of tyrosine phosphorylated pp125 FAK and paxillin has been found in cells transformed by Rous sarcoma virus [17] and in cells during embryogenesis [18]. Different sti...

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Section: Discussionmentioning

confidence: 99%

Echistatin inhibits pp125^FAK autophosphorylation, paxillin phosphorylation and pp125^FAK–paxillin interaction in fibronectin‐adherent melanoma cells

Morte¹,

Squillacioti²,

Garbi³

et al. 2000

European Journal of Biochemistry

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“…Other sequences in the ␤ 3 subunit, ␤ 3 -(214 -218) (RNRDA) and ␤ 3 -(217-231) (DAPEGGFDAIMQATV), may represent additional binding sites (37). The cross-linking site of disintegrins echistatin and eristostatin is located within ␤ 3 -(214 -302) (38). Using synthetic peptides, Steiner et al (39) presented evidence that the RNRDA motif plays a role in the expression of LIBS epitope.…”

Section: Table I the Effect Of Recombinant Echistatin Eristostatin mentioning

confidence: 99%

Structural Requirements of Echistatin for the Recognition of αvβ3 and α5β1Integrins

Wierzbicka-Patynowski¹,

Niewiarowski²,

Marcinkiewicz³

et al. 1999

Journal of Biological Chemistry

135

123

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There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of ␣ IIb ␤ 3 , ␣ v ␤ 3 and ␣ 5 ␤ 1 , and eristostatin, a similar disintegrin selectively inhibiting ␣ IIb ␤ 3 . In order to identify echistatin motifs required for selective recognition of ␣ v ␤ 3 and ␣ 5 ␤ 1 integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The integrins are a family of cell surface glycoproteins that act as receptors for extracellular matrix (ECM) 1 proteins, or for membrane-bound counter-receptors on other cells. Each integrin is a heterodimer that contains an ␣ and a ␤ subunit, the pairing of which specifies the ligand binding abilities of integrins (1). Integrins can bind adhesive ligands and upon this binding undergo conformational changes leading to the exposure of neoepitopes recognized by specific monoclonal antibodies called anti-ligand-induced binding site (LIBS) antibodies (2).The Arg-Gly-Asp (RGD) sequence is the cell attachment site of a large number of adhesive ECM, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands (3). In addition to the RGD motif, many adhesive receptors recognize other integrin-binding domains, such as the KQAGDV sequence (4) of fibrinogen ␥-chain, ILDV sequence of the CS1 region of fibronectin (5, 6), and RRETAWA sequence identified from a phage display library (7). It is known that the Arg and Asp residues are necessary but not sufficient to ensure binding activity. The RGD sequence is generally found to be associated with a series of probable ␤-bends, which result in a highly ordered structure. This highly ordered conformation might form the basis of the specific binding of many proteins containing this cell surface recognition sequence. Additional determinants of integrin specificity and the high affinity of RGDcontaining adhesive proteins for integrins may be the result of the specific conformation of the RGD sequence or by the amino acids immediately adjacent to the RGD site, creating an extended RGD locus.Disintegrins are snake venom proteins capable of binding to integrins and interfering with integrin function (8 -10). Disintegrins typically have an RGD sequence as their active site, except for barbourin containing a KGD sequence (11), and a new class of heterodimeric disintegrins such as EC3 and EMF10, containing MLD, VGD, and other recognition motifs (12). It appears that low molecular weight disintegrins, binding to integrins with an affinity approaching that of monoclonal antibodies, may represent a useful probe to identify functionally important motifs in the cell adhesion receptors. Scarborough et al. (13) postulated that the amino acid residue Cterminal to the RGD sequence determines selectivity of disintegrin for an integrin re...

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“…Early attempts to elucidate the nature of the bimolecular interaction between the β 3 ‐containing integrins, α V β 3 and α IIb β 3 , and radioiodinated RGD‐containing ligands by cross‐linking methods employed either homobifunctional chemical cross‐linkers (24) or arylazide‐based photoaffinity agents (25, 26). Although the predominant cross‐linking occurred with the β 3 subunit, only large segments within the receptor, such as the sequences 109–171 (24) or 217–302 (26) in α IIb β 3 and 61–203 in α V β 3 (25) could be assigned.…”

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confidence: 99%

Design and evaluation of benzophenone‐containing conformationally constrained ligands as tools for photoaffinity scanning of the integrin α_Vβ₃–ligand bimolecular interaction

Bitan

Scheibler

Teng

et al. 2000

The Journal of Peptide Research

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Integrins are cell-surface adhesion molecules involved in mediating cell-extracellular matrix interactions. High-resolution structural data are not available for these heterodimeric receptors. In order to generate tools for photoaffinity scanning of the RGD-binding site of human integrin alphaVbeta3. new conformationally constrained ligands were designed. The ligands were based on five different cyclic peptidic or peptidomimetic scaffolds with high affinity for alphaVbeta3. A single photoreactive group (a benzophenone moiety) was introduced at different positions relative to the RGD triad. In addition, an 125I or a biotin group was introduced as a reporting tag. Twenty-four cyclic ligands were prepared and their binding affinity for alphaVbeta3 was determined. In most cases, the modifications resulted in a 5- to 500-fold decrease in affinity relative to the unmodified scaffold. Analogs representing three of the five families were screened for their cross-linking efficiency. Ligands with submicromolar affinities cross-linked efficiently and specifically to the integrin receptor, whereas ligands with weaker affinities gave specific cross-linking, but with lower efficiency. Almost all of the screened ligands cross-linked predominantly to the beta3 subunit.

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Characterization of the Cross-Linking Site of Disintegrins Albolabrin, Bitistatin, Echistatin, and Eristostatin on Isolated Human Platelet Integrin GpIIb/IIIa

Cited by 24 publications

References 0 publications

Echistatin inhibits pp125^FAK autophosphorylation, paxillin phosphorylation and pp125^FAK–paxillin interaction in fibronectin‐adherent melanoma cells

Echistatin inhibits pp125^FAK autophosphorylation, paxillin phosphorylation and pp125^FAK–paxillin interaction in fibronectin‐adherent melanoma cells

Structural Requirements of Echistatin for the Recognition of αvβ3 and α5β1Integrins

Design and evaluation of benzophenone‐containing conformationally constrained ligands as tools for photoaffinity scanning of the integrin α_Vβ₃–ligand bimolecular interaction

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Characterization of the Cross-Linking Site of Disintegrins Albolabrin, Bitistatin, Echistatin, and Eristostatin on Isolated Human Platelet Integrin GpIIb/IIIa

Cited by 24 publications

References 0 publications

Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK–paxillin interaction in fibronectin‐adherent melanoma cells

Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK–paxillin interaction in fibronectin‐adherent melanoma cells

Structural Requirements of Echistatin for the Recognition of αvβ3 and α5β1Integrins

Design and evaluation of benzophenone‐containing conformationally constrained ligands as tools for photoaffinity scanning of the integrin αVβ3–ligand bimolecular interaction

Contact Info

Product

Resources

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Echistatin inhibits pp125^FAK autophosphorylation, paxillin phosphorylation and pp125^FAK–paxillin interaction in fibronectin‐adherent melanoma cells

Echistatin inhibits pp125^FAK autophosphorylation, paxillin phosphorylation and pp125^FAK–paxillin interaction in fibronectin‐adherent melanoma cells

Design and evaluation of benzophenone‐containing conformationally constrained ligands as tools for photoaffinity scanning of the integrin α_Vβ₃–ligand bimolecular interaction