The Type III Connecting Segment of Fibronectin Contains an Aspartic Acid Residue that Regulates the Rate of Binding to Integrin α<sub>4</sub>β<sub>1</sub>

Jongewaard, Ian N.; Tsai, Pamela M.; Smith, Jeffrey W.

doi:10.3109/15419069609081025

Cited by 18 publications

(12 citation statements)

References 39 publications

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“…The KLDAPT sequence reported here fits well into this pattern and shows that LDA is a novel recognition motif for activated ␣4␤1 and ␣4␤7, present in the FN-III5 repeat. In agreement with this, a recent report (27) has shown that mutants of IIICS in which the wild-type sequence EILDVP was changed to EILDAP, supported adhesion of CHO-␣4 transfected cells almost as effectively as the native sequence. This group also reported that increasing the time of the assay could overcome the impaired adhesion observed for certain mutants.…”

Section: Discussionsupporting

confidence: 81%

Fibronectin Type III5 Repeat Contains a Novel Cell Adhesion Sequence, KLDAPT, Which Binds Activated α4β1 and α4β7 Integrins

Moyano¹,

Bárbara²,

Domínguez‐Jiménez³

et al. 1997

Journal of Biological Chemistry

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The region of fibronectin encompassing type III repeats 4 -6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact Fibronectin (Fn) 1 is an extracellular matrix and plasma protein composed of structural domains that contain binding sites for other macromolecules (fibrin, heparin/proteoglycans, collagen), as well as for cells (reviewed in Hynes (1)). The NH 2 -terminal heparin binding domain or Hep I (see Fig. 1) also interacts with cell surfaces by binding to uncharacterized molecules and is mainly involved in formation of Fn matrices (1, 2). The Hep II domain displays the highest avidity for heparin and contains specific sequences which bind proteoglycans and/or the ␣4␤1 integrin at the cell surface and induce cell adhesion (3-5). One of these sites is H1, which is a ligand for ␣4␤1 in melanoma (6) and lymphoid cells (7). Two other sequences, CS-1 and CS-5, located within the IIICS segment (outside the Hep II domain) are also ligands for ␣4␤1 (8 -11) and bind with different affinities (12). These previous studies have established that ␣4␤1 may bind several sequences in the COOHterminal region of Fn and that these interactions are particularly important in lymphoid cells, where ␣4␤1 is highly expressed.The central Hep III domain is less well studied because it binds heparin only at low salt concentration, and the physiological significance of this interaction is unclear. Using DNA affinity chromatography, we and others previously isolated proteolytic fragments from this region of Fn, which displayed low affinity binding to heparin. These include a 14-kDa fragment corresponding to FN-III1 repeat (13) and two fragments of 18 -20 kDa (FN-III4-1/2 5 repeats) (14) and 30 kDa (FN-III4 -6 repeats) (15) from human and bovine plasma Fn, respectively. We also previously reported that an 80-kDa tryptic fragment (FN-III4-1/2 FN-III11) binds heparin with low avidity (16). It is not known whether this heparin binding domain also interacts with cells.The present study was undertaken to further characterize the properties of the Hep III domain of Fn. To this effect, we have prepared recombinant fragments encompassing type III homology repeats from this region and have studied their cell binding activities. We show that a fragment containing the FN-III5 repeat mediates adhesion of T and B lymphoid cells efficiently. Furthermore, we have identified a novel 6-amino acid-sequence as the active site in FN-III5, and we show that activated ␣4␤1 and ␣4␤7 integrins are receptors for this sequence and the FN-III5 fragment. These results therefore establish a novel function for the Hep III Fn domain.

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Section: Discussionsupporting

confidence: 81%

Fibronectin Type III5 Repeat Contains a Novel Cell Adhesion Sequence, KLDAPT, Which Binds Activated α4β1 and α4β7 Integrins

Moyano¹,

Bárbara²,

Domínguez‐Jiménez³

et al. 1997

Journal of Biological Chemistry

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“…Bacterial expression vectors for glutathione S-transferase (GST) fusion proteins encompassing the human FN CS-1 region and FN repeats 9 through 11 (9-11) were provided by J. W. Smith (Burnham Institute, La Jolla, CA) and J. W. Ramos (Rutgers University, New Brunswick, NJ), respectively, and were purified as described previously (18,38). VCAM-immunoglobulin (Ig), consisting of the seven extracellular Ig domains of VCAM-1 fused to the heavy chain of human IgG1, was expressed and purified as described previously (41).…”

Section: Methodsmentioning

confidence: 99%

Integrin α4β1 Promotes Focal Adhesion Kinase-Independent Cell Motility via α4 Cytoplasmic Domain-Specific Activation of c-Src

Hsia

Lim

Bernard‐Trifilo

et al. 2005

Molecular and Cellular Biology

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The fibronectin binding integrins ␣5␤1 and ␣4␤1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For ␣5␤1, ␤1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for ␣5␤1-stimulated cell motility and that exogenous expression of human ␣4 in FAK-null fibroblasts forms a functional ␣4␤1 receptor that promotes robust cell motility equal to the ␣5␤1 stimulation of wild-type and FAK-reconstituted fibroblasts. ␣4␤1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the ␣4 cytoplasmic domain, independent of direct paxillin binding to ␣4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. ␣4 cytoplasmic domain-initiated signaling led to a ϳ4-fold activation of c-Src which did not require paxillin binding to ␣4. Notably, ␣4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase ␣ overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. ␣4␤1-stimulated cell motility of triple-null Src ؊/؊ , c-Yes ؊/؊ , and Fyn ؊/؊ fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for ␣5␤1-stimulated FAK activation, our results support the existence of a novel ␣4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.Integrins are a family of heterodimeric ␣/␤ transmembrane cell adhesion receptors that play important roles in the regulation of cell migration during development, wound healing, inflammation, and the spread of tumor cells. Integrins do not possess intrinsic catalytic activity, and thus, signaling events are mediated by either lateral association with other receptors (8,52) or the clustering of signaling proteins with integrin cytoplasmic domains (44). As the composition of integrin signaling complexes is diverse and remains poorly defined (16), it is important to identify the molecular signaling signature of integrins that share a common ␤ subunit, bind to a common substrate such as fibronectin (FN), and function to promote cell motility. The FN binding integrins ␣5␤1 and ␣4␤1 share these properties.␣5␤1 is considered to be the classical FN receptor, with binding occurring within FN repeats III-9 and III-10 (33, 37). Rapid activation of protein-tyrosine kinases (PTKs) is one of the first signaling events associated with ␣5␤1 binding to FN, and signals generated by the ␤1 cytoplasmic domain are important in promoting cell motility (12, 39). Focal adhesion kinase (FAK) is recruited to sites of ␣5␤1 clustering through FAK C-terminal domain interactions with ␤1-integrin binding proteins such as talin and adaptor proteins such as paxillin (34). FN-stimulated FAK activation results in increased F...

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“…18 Collagen, ␣5, and ␣2 integrin antibodies (BD Biosciences), HP2/1 ␣4 integrin antibodies (Immunotech), and rat ␣-mouse CD31 antibodies (Invitrogen) were purchased. PS␣4 monoclonal antibodies to Ser 988 -phospho-␣4 integrin were as described.…”

Section: Complementary Dnas and Other Reagentsmentioning

confidence: 99%

Localized α4 Integrin Phosphorylation Directs Shear Stress–Induced Endothelial Cell Alignment

Goldfinger

Tzima

Stockton

et al. 2008

Circulation Research

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Abstract-Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to atheroprotection. Here we report that localized ␣4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress-induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial ␣4 integrins became phosphorylated on Ser 988 . In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced ␣4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a nonphosphorylatable ␣4(S 988 A) mutation failed to align in response to shear stress, thus establishing ␣4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent ␣4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress. (Circ Res. 2008;103:177-185.)

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The Type III Connecting Segment of Fibronectin Contains an Aspartic Acid Residue that Regulates the Rate of Binding to Integrin α₄β₁

Cited by 18 publications

References 39 publications

Fibronectin Type III5 Repeat Contains a Novel Cell Adhesion Sequence, KLDAPT, Which Binds Activated α4β1 and α4β7 Integrins

Fibronectin Type III5 Repeat Contains a Novel Cell Adhesion Sequence, KLDAPT, Which Binds Activated α4β1 and α4β7 Integrins

Integrin α4β1 Promotes Focal Adhesion Kinase-Independent Cell Motility via α4 Cytoplasmic Domain-Specific Activation of c-Src

Localized α4 Integrin Phosphorylation Directs Shear Stress–Induced Endothelial Cell Alignment

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The Type III Connecting Segment of Fibronectin Contains an Aspartic Acid Residue that Regulates the Rate of Binding to Integrin α4β1

Cited by 18 publications

References 39 publications

Fibronectin Type III5 Repeat Contains a Novel Cell Adhesion Sequence, KLDAPT, Which Binds Activated α4β1 and α4β7 Integrins

Fibronectin Type III5 Repeat Contains a Novel Cell Adhesion Sequence, KLDAPT, Which Binds Activated α4β1 and α4β7 Integrins

Integrin α4β1 Promotes Focal Adhesion Kinase-Independent Cell Motility via α4 Cytoplasmic Domain-Specific Activation of c-Src

Localized α4 Integrin Phosphorylation Directs Shear Stress–Induced Endothelial Cell Alignment

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The Type III Connecting Segment of Fibronectin Contains an Aspartic Acid Residue that Regulates the Rate of Binding to Integrin α₄β₁