H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 92
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The 20 or soisoforms of fibronectin are generated by alternative splicingof the RNA transcript produced from a single gene (see Figure 4-15). Fibronectins are essential for the migration anddifferentiation of many cell types in embryogenesis. Theseproteins are also important for wound healing because theypromote blood clotting and facilitate the migration ofmacrophages and other immune cells into the affected area.Fibronectins help attach cells to the extracellular matrixby binding to other ECM components, particularly fibrouscollagens and heparan sulfate proteoglycans, and to cellsurface adhesion receptors such as integrins (see Figure 6-2).Through their interactions with adhesion receptors (e.g.,51 integrin), fibronectins influence the shape and movement of cells and the organization of the cytoskeleton.
Conversely, by regulating their receptor-mediated attachmentsto fibronectin and other ECM components, cells can sculptthe immediate ECM environment to suit their needs.Fibronectins are dimers of two similar polypeptideslinked at their C-termini by two disulfide bonds; each chainis about 60–70 nm long and 2–3 nm thick. Partial digestion2216.4 • The Extracellular Matrix of Nonepithelial TissuesFibrin,heparansulfate–bindingrepeatsCollagenbindingrepeatsHeparansulfate–bindingrepeatIntegrinbindingrepeatsFibrinbindingrepeatsCOOHNH2EIIIBRepeating amino acid sequences:Type IType IIEIIIAIIICSType III▲ FIGURE 6-23 Organization of fibronectin chains.
Only oneof the two chains present in the dimeric fibronectin molecule isshown; both chains have very similar sequences. Each chaincontains about 2446 amino acids and is composed of three typesof repeating amino acid sequences. Circulating fibronectin lacksone or both of the type III repeats designated EIIIA and EIIIBowing to alternative mRNA splicing (see Figure 4-15). At leastfive different sequences may be present in the IIICS region as aof fibronectin with low amounts of proteases and analysisof the fragments showed that each chain comprises six functional regions with different ligand-binding specificities (Figure 6-23).
Each region, in turn, contains multiple copies ofcertain sequences that can be classified into one of threetypes. These classifications are designated fibronectin type I,II, and III repeats, on the basis of similarities in amino acidsequence, although the sequences of any two repeats of agiven type are not always identical. These linked repeats givethe molecule the appearance of beads on a string. The combination of different repeats composing the regions, anotherexample of combinatorial diversity, confers on fibronectin itsability to bind multiple ligands.S SS SCOOHresult of alternative splicing.
Each chain contains six domains (tanboxes), some of which contain specific binding sites for heparansulfate, fibrin (a major constituent of blood clots), collagen, andcell-surface integrins. The integrin-binding domain is also knownas the cell-binding domain. [Adapted from G. Paolella, M. Barone,and F. Baralle, 1993, in M.
Zern and L. Reid, eds., Extracellular Matrix,Marcel Dekker, pp. 3–24.]One of the type III repeats in the cell-binding region of fibronectin mediates binding to certain integrins. The resultsof studies with synthetic peptides corresponding to parts ofthis repeat identified the tripeptide sequence Arg-Gly-Asp,usually called the RGD sequence, as the minimal sequencewithin this repeat required for recognition by those integrins.In one study, heptapeptides containing the RGD sequence ora variation of this sequence were tested for their ability tomediate the adhesion of rat kidney cells to a culture dish.
Theresults showed that heptapeptides containing the RGDsequence mimicked intact fibronectin’s ability to stimulateintegrin-mediated adhesion, whereas variant heptapeptideslacking this sequence were ineffective (Figure 6-24).Relative amounts of bound cells (stain intensity)1.4 EXPERIMENTAL FIGURE 6-24 A specific tripeptideGRGDSPC1.2GRGDAPCPRGDVDC1.0YKPGEGKRGDACEGDSG0.80.60.4GRADSPCGRGESPCGKGDSPCDREDSRC0.21101001000Peptide concentration (nmol/ml)sequence (RGD) in the cell-binding region of fibronectin isrequired for adhesion of cells. The cell-binding region offibronectin contains an integrin-binding heptapeptide sequence,GRDSPC in the single-letter amino acid code (see Figure 2-13).This heptapeptide and several variants were synthesizedchemically.
Different concentrations of each synthetic peptidewere added to polystyrene dishes that had the proteinimmunoglobulin G (IgG) firmly attached to their surfaces; thepeptides were then chemically cross-linked to the IgG.Subsequently, cultured normal rat kidney cells were added to thedishes and incubated for 30 minutes to allow adhesion. After thenonbound cells were washed away, the relative amounts of cellsthat had adhered firmly were determined by staining the boundcells with a dye and measuring the intensity of the staining witha spectrophotometer. The plots shown here indicate that celladhesion increased above the background level with increasingpeptide concentration for those peptides containing the RGDsequence but not for the variants lacking this sequence(modification underlined).
[From M. D. Pierschbacher and E. Ruoslahti,1984, Proc. Nat’l. Acad. Sci. USA 81:5985.]222CHAPTER 6 • Integrating Cells into Tissues(a)Fibrin,heparansulfatebinding(b)CollagenbindingEIIIBEIIIAIIICSRGDSSNH2COOHHeparansulfatebindingType I repeatFibrinbindingSynergyregionRGDsequenceType II repeatType III repeatIntegrin▲ FIGURE 6-25 Model of fibronectin binding to integrinthrough its RGD-containing type III repeat. (a) Scale modelof fibronectin is shown docked by two type III repeats to theextracellular domains of integrin. Structures of fibronectin’sdomains 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 byA three-dimensional model of fibronectin binding tointegrin based on structures of parts of both fibronectinand integrin has been assembled (Figure 6-25a).
In a highresolution structure of the integrin-binding fibronectin typeIII repeat and its neighboring type III domain, the RGD sequence is at the apex of a loop that protrudes outward fromthe molecule, in a position facilitating binding to integrins(Figure 6-25a, b). Although the RGD sequence is requiredfor binding to several integrins, its affinity for integrins issubstantially 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., partsof adjacent repeats, such as the synergy region; see Figurearrows. (b) A high-resolution structure shows that the RGDbinding sequence (red) extends outward in a loop from itscompact type III domain on the same side of fibronectin as thesynergy region (blue), which also contributes to high-affinitybinding to integrins.
[Adapted from D. J. Leahy et al., 1996, Cell84:161.]6-25b) and in other RGD-containing proteins enhance theirbinding to certain integrins. Moreover, the simple solubledimeric forms of fibronectin produced by the liver or fibroblasts are initially in a nonfunctional closed conformationthat binds poorly to integrins because the RGD sequence isnot readily accessible. The adsorption of fibronectin to a col(a) EXPERIMENTAL FIGURE 6-26 Integrins mediate linkagebetween fibronectin in the extracellular matrix and thecytoskeleton. (a) Immunofluorescent micrograph of a fixedcultured fibroblast showing colocalization of the 51 integrinand actin-containing stress fibers. The cell was incubated withtwo types of monoclonal antibody: an integrin-specific antibodylinked to a green fluorescing dye and an actin-specific antibodylinked to a red fluorescing dye.
Stress fibers are long bundles ofactin microfilaments that radiate inward from points where thecell contacts a substratum. At the distal end of these fibers, nearthe plasma membrane, the coincidence of actin (red) andfibronectin-binding integrin (green) produces a yellowfluorescence. (b) Electron micrograph of the junction offibronectin and actin fibers in a cultured fibroblast. Individualactin-containing 7-nm microfilaments, components of a stressfiber, end at the obliquely sectioned cell membrane.
Themicrofilaments appear in close proximity to the thicker, denselystained fibronectin fibrils on the outside of the cell. [Part (a) fromJ. 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.](b)FibronectinfibrilsCellexteriorPlasmamembraneActin-containingmicrofilamentsCell interior0.5 m6.5 • Adhesive Interactions and Nonepithelial Cellslagen matrix or the basal lamina or, experimentally, to a plastic tissue-culture dish results in a conformational change thatenhances 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 ofintegrins in cross-linking fibronectin and other ECM components to the cytoskeleton.
For example, the colocalization ofcytoskeletal actin filaments and integrins within cells can bevisualized by fluorescence microscopy (Figure 6-26a). Thebinding of cell-surface integrins to fibronectin in the matrixinduces the actin cytoskeleton–dependent movement of someintegrin 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 stretchesthe fibronectin. This stretching promotes self-association ofthe fibronectin into multimeric fibrils.The force needed to unfold and expose functional selfassociation sites in fibronectin is much less than thatneeded to disrupt fibronectin–integrin binding. Thus fibronectin molecules remain bound to integrin while cellgenerated mechanical forces induce fibril formation.
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