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A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly.

Sechler JL, Rao H, Cumiskey AM, Vega-Colón I, Smith MS, Murata T, Schwarzbauer JE - J. Cell Biol. (2001)

Bottom Line: In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded.Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site.Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

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Assembly of FNrIII4–5/9–10. CHOα5 cells were cultured in the presence of 50 μg/ml FNrIII4–5/9–10 for 4 (A) and 16 (B) h. (C) DOC-insoluble material was isolated from CHOα5 cells 4 and 16 h after incubation with 50 μg/ml FNrIII4–5/9–10 and analyzed as in Fig. 3. Locations of high molecular mass multimers are indicated with arrow and bracket. 180-kD molecular mass standard is indicated by dash. Bar, 10 μm.
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fig8: Assembly of FNrIII4–5/9–10. CHOα5 cells were cultured in the presence of 50 μg/ml FNrIII4–5/9–10 for 4 (A) and 16 (B) h. (C) DOC-insoluble material was isolated from CHOα5 cells 4 and 16 h after incubation with 50 μg/ml FNrIII4–5/9–10 and analyzed as in Fig. 3. Locations of high molecular mass multimers are indicated with arrow and bracket. 180-kD molecular mass standard is indicated by dash. Bar, 10 μm.

Mentions: The cell binding domain consisting of an RGD sequence and synergy site in repeats III9–10 is essential for initiation of FN matrix assembly by α5β1 integrin (Sechler et al., 1996, 1997). Deletions within repeats III1–7 position the cell binding domain closer to the NH2-terminal assembly domain and could affect recFN fibril formation. To eliminate the possibility that changes in the domain organization can alter FN assembly, we created FNrIII4–5/9–10. Repeats III4–5 were replaced with III9–10 and the RGD sequence was deleted from its native position within the cell binding domain (Fig. 1). Therefore, the only functional III9–10 pair is in the position normally occupied by III4–5. CHOα5 cells efficiently assembled FNrIII4–5/9–10 into a DOC-insoluble fibrillar matrix identical to that of native FN at all time points (Fig. 8 , A–C). Cell cycle progression by CHOα5 cells assembling either FN or FNrIII4–5/9–10 was identical, as were the levels of focal adhesion kinase phosphorylation (unpublished data). These results demonstrate that the location of the cell binding domain is not restricted to the center of the molecule and that displacement toward the NH2 terminus does not reduce FN function in matrix assembly or its ability to influence cell cycle progression.


A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly.

Sechler JL, Rao H, Cumiskey AM, Vega-Colón I, Smith MS, Murata T, Schwarzbauer JE - J. Cell Biol. (2001)

Assembly of FNrIII4–5/9–10. CHOα5 cells were cultured in the presence of 50 μg/ml FNrIII4–5/9–10 for 4 (A) and 16 (B) h. (C) DOC-insoluble material was isolated from CHOα5 cells 4 and 16 h after incubation with 50 μg/ml FNrIII4–5/9–10 and analyzed as in Fig. 3. Locations of high molecular mass multimers are indicated with arrow and bracket. 180-kD molecular mass standard is indicated by dash. Bar, 10 μm.
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Related In: Results  -  Collection

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fig8: Assembly of FNrIII4–5/9–10. CHOα5 cells were cultured in the presence of 50 μg/ml FNrIII4–5/9–10 for 4 (A) and 16 (B) h. (C) DOC-insoluble material was isolated from CHOα5 cells 4 and 16 h after incubation with 50 μg/ml FNrIII4–5/9–10 and analyzed as in Fig. 3. Locations of high molecular mass multimers are indicated with arrow and bracket. 180-kD molecular mass standard is indicated by dash. Bar, 10 μm.
Mentions: The cell binding domain consisting of an RGD sequence and synergy site in repeats III9–10 is essential for initiation of FN matrix assembly by α5β1 integrin (Sechler et al., 1996, 1997). Deletions within repeats III1–7 position the cell binding domain closer to the NH2-terminal assembly domain and could affect recFN fibril formation. To eliminate the possibility that changes in the domain organization can alter FN assembly, we created FNrIII4–5/9–10. Repeats III4–5 were replaced with III9–10 and the RGD sequence was deleted from its native position within the cell binding domain (Fig. 1). Therefore, the only functional III9–10 pair is in the position normally occupied by III4–5. CHOα5 cells efficiently assembled FNrIII4–5/9–10 into a DOC-insoluble fibrillar matrix identical to that of native FN at all time points (Fig. 8 , A–C). Cell cycle progression by CHOα5 cells assembling either FN or FNrIII4–5/9–10 was identical, as were the levels of focal adhesion kinase phosphorylation (unpublished data). These results demonstrate that the location of the cell binding domain is not restricted to the center of the molecule and that displacement toward the NH2 terminus does not reduce FN function in matrix assembly or its ability to influence cell cycle progression.

Bottom Line: In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded.Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site.Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

Show MeSH