Rgd

Type I repeat Type II repeat Type III repeat

Heparan Fibrin sulfate binding binding

Integrin

Heparan Fibrin sulfate binding binding

SS COOH

Synergy region V

RGD sequence

Integrin

▲ FIGURE 6-25 Model of fibronectin binding to integrin through its RGD-containing type III repeat. (a) Scale model of fibronectin is shown docked by two type III repeats to the extracellular domains of integrin. Structures of fibronectin's domains were determined from fragments of the molecule. The EIIIA, EIIIB, and IIICS domains (not shown; see Figure 6-23) are variably spliced into the structure at locations indicated by

A three-dimensional model of fibronectin binding to integrin based on structures of parts of both fibronectin and integrin has been assembled (Figure 6-25a). In a highresolution structure of the integrin-binding fibronectin type III repeat and its neighboring type III domain, the RGD sequence is at the apex of a loop that protrudes outward from the molecule, in a position facilitating binding to integrins (Figure 6-25a, b). Although the RGD sequence is required for binding to several integrins, its affinity for integrins is substantially less than that of intact fibronectin or of the entire cell-binding region in fibronectin. Thus structural features near to the RGD sequence in fibronectins (e.g., parts of adjacent repeats, such as the synergy region; see Figure

► EXPERIMENTAL FIGURE 6-26 Integrins mediate linkage between fibronectin in the extracellular matrix and the cytoskeleton. (a) Immunofluorescent micrograph of a fixed cultured fibroblast showing colocalization of the a5pi integrin and actin-containing stress fibers. The cell was incubated with two types of monoclonal antibody: an integrin-specific antibody linked to a green fluorescing dye and an actin-specific antibody linked to a red fluorescing dye. Stress fibers are long bundles of actin microfilaments that radiate inward from points where the cell contacts a substratum. At the distal end of these fibers, near the plasma membrane, the coincidence of actin (red) and fibronectin-binding integrin (green) produces a yellow fluorescence. (b) Electron micrograph of the junction of fibronectin and actin fibers in a cultured fibroblast. Individual actin-containing 7-nm microfilaments, components of a stress fiber, end at the obliquely sectioned cell membrane. The microfilaments appear in close proximity to the thicker, densely stained fibronectin fibrils on the outside of the cell. [Part (a) from J. Duband et al., 1988, J. Cell Biol. 107:1385. Part (b) from I. J. Singer, 1979, Cell 16:675; courtesy of I. J. Singer; copyright 1979, MIT]

arrows. (b) A high-resolution structure shows that the RGD binding sequence (red) extends outward in a loop from its compact type III domain on the same side of fibronectin as the synergy region (blue), which also contributes to high-affinity binding to integrins. [Adapted from D. J. Leahy et al., 1996, Cell 84:161.]

6-25b) and in other RGD-containing proteins enhance their binding to certain integrins. Moreover, the simple soluble dimeric forms of fibronectin produced by the liver or fibro-blasts are initially in a nonfunctional closed conformation that binds poorly to integrins because the RGD sequence is not readily accessible. The adsorption of fibronectin to a col

lagen matrix or the basal lamina or, experimentally, to a plastic tissue-culture dish results in a conformational change that enhances its ability to bind to cells. Most likely, this conformational change increases the accessibility of the RGD sequence for integrin binding.

Microscopy and other experimental approaches (e.g., biochemical binding experiments) have demonstrated the role of integrins in cross-linking fibronectin and other ECM components to the cytoskeleton. For example, the colocalization of cytoskeletal actin filaments and integrins within cells can be visualized by fluorescence microscopy (Figure 6-26a). The binding of cell-surface integrins to fibronectin in the matrix induces the actin cytoskeleton-dependent movement of some integrin molecules in the plane of the membrane. The ensuing mechanical tension due to the relative movement of different integrins bound to a single fibronectin dimer stretches the fibronectin. This stretching promotes self-association of the fibronectin into multimeric fibrils.

The force needed to unfold and expose functional self-association sites in fibronectin is much less than that needed to disrupt fibronectin-integrin binding. Thus fi-bronectin molecules remain bound to integrin while cell-generated mechanical forces induce fibril formation. In effect, the integrins through adapter proteins transmit the in-tracellular forces generated by the actin cytoskeleton to extracellular fibronectin. Gradually, the initially formed fibronectin fibrils mature into highly stable matrix components by covalent cross-linking. In some electron micrographic images, exterior fibronectin fibrils appear to be aligned in a seemingly continuous line with bundles of actin fibers within the cell (Figure 6-26b). These observations and the results from other studies provided the first example of a molecularly well defined adhesion receptor (i.e., an inte-grin) forming a bridge between the intracellular cytoskeleton and the extracellular matrix components—a phenomenon now known to be widespread.

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