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Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment.

Manabe R, Ohe N, Maeda T, Fukuda T, Sekiguchi K - J. Cell Biol. (1997)

Bottom Line: To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins.Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module.Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, Osaka Medical Center for Maternal and Child Health, Japan.

ABSTRACT
Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA- FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin alpha5 and beta1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 bound more avidly to EDA+ FN than to EDA- FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA- FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin alpha5beta1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

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Binding of integrin α5β1 to  FN fragments. (A) Microtiter plates  were coated with 20 μg/ml of rFN(C)  (open bars) or rFN(AC) (closed bars)  that had been digested with thermolysin for 0 min (Undigested) and 10 min  (Digested) as described in Materials  and Methods. The plates were incubated with integrin α5β1 reconstituted  in phosphatidylcholine liposomes for 6 h  at room temperature. Quantities of  bound integrin α5β1 liposomes were  expressed as percentages of the total input radioactivity after subtraction of  the radioactivity bound to plates coated  only with BSA. Each bar represents the  mean ± SD (n = 3). (B) Microtiter  plates were coated with 20 μg/ml (open bars) or 80 μg/ml (closed and hatched bars) of GST fusion proteins containing the CCBD alone  (GST-C) or both the CCBD and the Hep2 domain with (GST-CAH) or without (GST-CH) the EDA segment. Integrin α5β1 liposomes  were added to the plates and incubated for 6 h at room temperature in the absence (open and closed bars) or presence (hatched bars) of  the anti-integrin α5 mAb 8F1 (10 μg/ml). Each bar represents the mean ± SD (n = 6).
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Figure 10: Binding of integrin α5β1 to FN fragments. (A) Microtiter plates were coated with 20 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) that had been digested with thermolysin for 0 min (Undigested) and 10 min (Digested) as described in Materials and Methods. The plates were incubated with integrin α5β1 reconstituted in phosphatidylcholine liposomes for 6 h at room temperature. Quantities of bound integrin α5β1 liposomes were expressed as percentages of the total input radioactivity after subtraction of the radioactivity bound to plates coated only with BSA. Each bar represents the mean ± SD (n = 3). (B) Microtiter plates were coated with 20 μg/ml (open bars) or 80 μg/ml (closed and hatched bars) of GST fusion proteins containing the CCBD alone (GST-C) or both the CCBD and the Hep2 domain with (GST-CAH) or without (GST-CH) the EDA segment. Integrin α5β1 liposomes were added to the plates and incubated for 6 h at room temperature in the absence (open and closed bars) or presence (hatched bars) of the anti-integrin α5 mAb 8F1 (10 μg/ml). Each bar represents the mean ± SD (n = 6).

Mentions: The EDA segment may alter the conformation of CCBD by two possible mechanisms. In one model, the EDA segment inserted between the III11 and III12 modules may induce steric distortion of its neighboring modules, i.e., III11 and III12, by readjusting the intermodular interfaces, which in turn affects the conformation of adjacent modules including RGD-containing III10. Alternatively, insertion of the EDA segment may alter the global conformation of the FN molecule by twisting the NH2-terminal two-thirds of the molecule. Adjacent type III modules have been shown to be interconnected with rotations along the long axis, often in a pseudotwofold relationship (Huber et al., 1994; Leahy et al., 1996). To test these two possibilities, we examined binding affinities of recombinant FNs for integrin α5β1 before and after limited proteolysis with thermolysin. Under the conditions employed (Sekiguchi et al., 1985), the central region of the FN molecule including CCBD and the adjacent Hep2 domain was released by thermolysin as 150–120-kD fragments from rFN(C) and as 160–130-kD fragments from rFN(AC) (data not shown). The integrin-binding activity of rFN(C) was significantly increased after limited proteolysis, reaching a level comparable to that of rFN(AC) after thermolysin digestion (Fig. 10 A). It was also noted that the integrin-binding activity of rFN(AC) was slightly decreased after limited proteolysis. These results do not fit with the first possibility based on the neighboring effects of the inserted EDA segment on the III10 module but rather support the second model that insertion of the EDA segment potentiates integrin binding to CCBD by altering the global conformation of the FN molecule, thereby either increasing integrin accessibility to CCBD or optimizing the local conformation of the RGD-containing III10 module by perturbing the constraints applied to CCBD.


Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment.

Manabe R, Ohe N, Maeda T, Fukuda T, Sekiguchi K - J. Cell Biol. (1997)

Binding of integrin α5β1 to  FN fragments. (A) Microtiter plates  were coated with 20 μg/ml of rFN(C)  (open bars) or rFN(AC) (closed bars)  that had been digested with thermolysin for 0 min (Undigested) and 10 min  (Digested) as described in Materials  and Methods. The plates were incubated with integrin α5β1 reconstituted  in phosphatidylcholine liposomes for 6 h  at room temperature. Quantities of  bound integrin α5β1 liposomes were  expressed as percentages of the total input radioactivity after subtraction of  the radioactivity bound to plates coated  only with BSA. Each bar represents the  mean ± SD (n = 3). (B) Microtiter  plates were coated with 20 μg/ml (open bars) or 80 μg/ml (closed and hatched bars) of GST fusion proteins containing the CCBD alone  (GST-C) or both the CCBD and the Hep2 domain with (GST-CAH) or without (GST-CH) the EDA segment. Integrin α5β1 liposomes  were added to the plates and incubated for 6 h at room temperature in the absence (open and closed bars) or presence (hatched bars) of  the anti-integrin α5 mAb 8F1 (10 μg/ml). Each bar represents the mean ± SD (n = 6).
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Figure 10: Binding of integrin α5β1 to FN fragments. (A) Microtiter plates were coated with 20 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) that had been digested with thermolysin for 0 min (Undigested) and 10 min (Digested) as described in Materials and Methods. The plates were incubated with integrin α5β1 reconstituted in phosphatidylcholine liposomes for 6 h at room temperature. Quantities of bound integrin α5β1 liposomes were expressed as percentages of the total input radioactivity after subtraction of the radioactivity bound to plates coated only with BSA. Each bar represents the mean ± SD (n = 3). (B) Microtiter plates were coated with 20 μg/ml (open bars) or 80 μg/ml (closed and hatched bars) of GST fusion proteins containing the CCBD alone (GST-C) or both the CCBD and the Hep2 domain with (GST-CAH) or without (GST-CH) the EDA segment. Integrin α5β1 liposomes were added to the plates and incubated for 6 h at room temperature in the absence (open and closed bars) or presence (hatched bars) of the anti-integrin α5 mAb 8F1 (10 μg/ml). Each bar represents the mean ± SD (n = 6).
Mentions: The EDA segment may alter the conformation of CCBD by two possible mechanisms. In one model, the EDA segment inserted between the III11 and III12 modules may induce steric distortion of its neighboring modules, i.e., III11 and III12, by readjusting the intermodular interfaces, which in turn affects the conformation of adjacent modules including RGD-containing III10. Alternatively, insertion of the EDA segment may alter the global conformation of the FN molecule by twisting the NH2-terminal two-thirds of the molecule. Adjacent type III modules have been shown to be interconnected with rotations along the long axis, often in a pseudotwofold relationship (Huber et al., 1994; Leahy et al., 1996). To test these two possibilities, we examined binding affinities of recombinant FNs for integrin α5β1 before and after limited proteolysis with thermolysin. Under the conditions employed (Sekiguchi et al., 1985), the central region of the FN molecule including CCBD and the adjacent Hep2 domain was released by thermolysin as 150–120-kD fragments from rFN(C) and as 160–130-kD fragments from rFN(AC) (data not shown). The integrin-binding activity of rFN(C) was significantly increased after limited proteolysis, reaching a level comparable to that of rFN(AC) after thermolysin digestion (Fig. 10 A). It was also noted that the integrin-binding activity of rFN(AC) was slightly decreased after limited proteolysis. These results do not fit with the first possibility based on the neighboring effects of the inserted EDA segment on the III10 module but rather support the second model that insertion of the EDA segment potentiates integrin binding to CCBD by altering the global conformation of the FN molecule, thereby either increasing integrin accessibility to CCBD or optimizing the local conformation of the RGD-containing III10 module by perturbing the constraints applied to CCBD.

Bottom Line: To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins.Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module.Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, Osaka Medical Center for Maternal and Child Health, Japan.

ABSTRACT
Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA- FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin alpha5 and beta1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 bound more avidly to EDA+ FN than to EDA- FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA- FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin alpha5beta1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

Show MeSH
Related in: MedlinePlus