Cryptic Self-association Sites in Type III Modules of Fibronectin

Abstract

In an effort to detect interactions between other Fn domains, all fragments were coupled to Sepharose, and each fragment was tested on each affinity matrix before and after denaturation. The only interaction detected was that of fluid phase III 1 with immobilized denatured 110-kDa CBF and 40-kDa Hep-2, both of which contain type III domains. Analysis of subfragments revealed this activity to be dominated by domains III 7 and III 15 . Fn itself did not bind to the denatured fragments. Thus, domain III 1 contains two cryptic "self-association sites," one that is buried in the core of the fold but recognizes many Fn fragments when presented as a peptide and another that is concealed in Fn but exposed in the native isolated domain and recognizes cryptic sites in two other type III domains. These interactions between type III domains could play an important role in assembly of Fn multimers in the extracellular matrix. Fibronectin (Fn)1 circulates in plasma as a 550-kDa 2-chain monomer that can be transformed by cultured fibroblasts into an insoluble fibrillar structure during the cell-driven process of matrix assembly (1). Several regions of Fn have been implicated in this process. N-terminal fragments containing the first five type I modules bind to cell layers and inhibit matrix assembly but are not themselves incorporated into the insoluble matrix unless bivalent, and then most efficiently in the presence of intact Fn (2-8). For a long time, it was thought that N-terminal fragments were interacting with a cell-surface "matrix assembly receptor" that was distinct from integrins that interact with Fn primarily via the Arg-Gly-Asp in the 10th type III domain. At least two unidentified molecules have been proposed as candidates for the matrix assembly receptor by virtue of their interaction with N-terminal fragments of Fn (9, 10). However, none has been further characterized or shown to play a role in matrix assembly. More recently, it is beginning to appear that the matrix assembly receptor, i.e. the molecule responsible for binding N-terminal fragments, might be Fn itself, perhaps conformationally altered by incorporation into the matrix (5, 11, 12).The fact that Fn matrix assembly could be inhibited with a monoclonal antibody directed to module III 1 (13) prompted efforts to examine the interaction of this module with Fn. Morla and Ruoslahti (14) showed that a synthetic 31-mer peptide with N-terminal sequence NAPQ . . . , derived from the middle of III 1 , was able to bind Fn, but the site of its interaction was not determined. The same group later reported that a longer recombinant peptide with the same N terminus, when incubated with whole Fn, was able to induce the formation of polymers that were stable in SDS in the absence of reducing agents and exhibited superior adhesive properties toward fibroblasts (15). At the same time, it was shown by Hocking et al.(11) that module III 1 , when adsorbed to plastic above its denaturation temperature, was able to bind Fn and its N-terminal 70-kDa fragment; other fragments...

Comparison of the early stages of forced unfolding for fibronectin type III modules

“…Cryptic sites have been specifically proposed to exist in FN-III 1 , FN-III [7][8] , and FN-III 10 (11). Although our SMD simulations suggest that FN-III 7 is relatively stable, the cryptic site in the FN-III 7-8 dimer could be located at the interface between the two modules (12), implying that prestretching of FN-III 8 into the aligned state exposes the interface between the modules. Other researchers have suggested that FN-III 10 contains a cryptic site that binds to FN-III 1 when FN-III 10 is denatured (7,39).…”

“…Forced unfolding of the FN-III 10 module also has been suggested to modify exposure of the RGD loop, thus influencing FNЈs accessibility to integrins (9). Mechanical forces, furthermore, are essential in initiating FN fibril assembly (10), potentially through exposure of cryptic sites, and͞or by swapping complementary ␤-strands of partially unfolded FN-III modules during refolding (11,12). The functional states of FN are tightly regulated because FN is central in mediating and regulating cell adhesion, proliferation, and migration through various integrindependent signaling pathways (5).…”

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

“…There are at least three mechanisms by which hyperextension of Fn can alter its functional states: (i) by exposing cryptic sites that are buried between Fn modules in the native state; (ii) by changing the relative distance of two or more binding sites that bind the same receptor; and (iii) by straightening the hydrophilic loops that carry binding sites (30). Cryptic sites have been identified on FnIII 1 , FnIII [7][8] , and FnIII 10 that must be exposed to initiate Fn matrix assembly (31)(32)(33)(34) (35). This reduction in integrin affinity is caused by an increase in the distance between the arginine-glycine-aspartic acid (RGD) loop and its synergy site, from 3.2 nm to 5.6 nm.…”

Fibronectin in aging extracellular matrix fibrils is progressively unfolded by cells and elicits an enhanced rigidity response

“…[29][30][31][32][33][34] Moreover, the exposure of cryptic sites in Fn modules is an important event regulating integrin binding, cell signaling and the ability of cells to construct new matrix fibrils. 30,[35][36][37] Conversely, changes in cell contractility have been shown to alter access to these same sites within matrix fibrils. Inhibition of Rho A, an agonist of cell contractility, prevents the exposure of a cryptic site in FnIII 1 , and disruption of the actin cytoskeleton with cytochalasin D causes refolding of Fn type III modules in fibrils assembled between cells and an underlying glass substrate.…”