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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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DOC-soluble and -insoluble FNΔIII1. CHOα5 cells were incubated with 50 μg/ml pFN or FNΔIII1 for the indicated times and lysed in buffered DOC. DOC-soluble (A) and -insoluble (B) fractions were separated in 5% polyacrylamide-SDS gels without reduction and transferred to nitrocellulose. FN was detected on immunoblots with monoclonal anti-FN antibody IC3 and chemiluminescence reagents. Dimeric pFN and FNΔIII1 are present (arrowhead) as well as high molecular mass multimers at the top of the stacking (bracket) and at the interface of the stacking and separating gels (arrow).
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fig3: DOC-soluble and -insoluble FNΔIII1. CHOα5 cells were incubated with 50 μg/ml pFN or FNΔIII1 for the indicated times and lysed in buffered DOC. DOC-soluble (A) and -insoluble (B) fractions were separated in 5% polyacrylamide-SDS gels without reduction and transferred to nitrocellulose. FN was detected on immunoblots with monoclonal anti-FN antibody IC3 and chemiluminescence reagents. Dimeric pFN and FNΔIII1 are present (arrowhead) as well as high molecular mass multimers at the top of the stacking (bracket) and at the interface of the stacking and separating gels (arrow).

Mentions: Biochemically, FNΔIII1 and native FN matrices were indistinguishable. Equivalent amounts of both proteins were associated with CHOα5 cells in the DOC-soluble fractions (Fig. 3 A). Similar proportions of DOC-insoluble material were formed from FN and FNΔIII1 during a 16-h incubation (Fig. 3 B). As has been shown previously for FN (McKeown-Longo and Mosher, 1983; Sechler et al., 1996), there was continued incorporation of FNΔIII1 into DOC-insoluble matrices and high molecular mass aggregates (Fig. 3 B, 48 h). These data show that III1 is neither required for the formation of fibrils, nor responsible for the altered rate of assembly observed with FNΔIII1–7.


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)

DOC-soluble and -insoluble FNΔIII1. CHOα5 cells were incubated with 50 μg/ml pFN or FNΔIII1 for the indicated times and lysed in buffered DOC. DOC-soluble (A) and -insoluble (B) fractions were separated in 5% polyacrylamide-SDS gels without reduction and transferred to nitrocellulose. FN was detected on immunoblots with monoclonal anti-FN antibody IC3 and chemiluminescence reagents. Dimeric pFN and FNΔIII1 are present (arrowhead) as well as high molecular mass multimers at the top of the stacking (bracket) and at the interface of the stacking and separating gels (arrow).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196193&req=5

fig3: DOC-soluble and -insoluble FNΔIII1. CHOα5 cells were incubated with 50 μg/ml pFN or FNΔIII1 for the indicated times and lysed in buffered DOC. DOC-soluble (A) and -insoluble (B) fractions were separated in 5% polyacrylamide-SDS gels without reduction and transferred to nitrocellulose. FN was detected on immunoblots with monoclonal anti-FN antibody IC3 and chemiluminescence reagents. Dimeric pFN and FNΔIII1 are present (arrowhead) as well as high molecular mass multimers at the top of the stacking (bracket) and at the interface of the stacking and separating gels (arrow).
Mentions: Biochemically, FNΔIII1 and native FN matrices were indistinguishable. Equivalent amounts of both proteins were associated with CHOα5 cells in the DOC-soluble fractions (Fig. 3 A). Similar proportions of DOC-insoluble material were formed from FN and FNΔIII1 during a 16-h incubation (Fig. 3 B). As has been shown previously for FN (McKeown-Longo and Mosher, 1983; Sechler et al., 1996), there was continued incorporation of FNΔIII1 into DOC-insoluble matrices and high molecular mass aggregates (Fig. 3 B, 48 h). These data show that III1 is neither required for the formation of fibrils, nor responsible for the altered rate of assembly observed with FNΔIII1–7.

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