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Assembly of fibronectin into extracellular matrix
Assembly of fibronectin
into extracellular
matrix
Deane F. Mosher University
of Wisconsin,
Madison,
USA
Assembly of fibronectin into a fibrillar matrix is part of a complicated process by which the intracelltilar cytoskeleton determines the pattern of assembly of extracellular matrix and vice versa. Assembly is restricted to dimeric fibronectin molecules with intact amino-terminal modules and occurs at specialized cell-surface assembly sites. Cellular display of assembly sites is labile and requires that cells be under tension and have functional & integrins. Assembly is described as a stepwise event in which protameric dimers bind to assembly sites, multimerization occurs, and assembly sites are regenerated. Current
Opinion
in Structural
1993, 3:214-222
is however of considerable biological importance and interesting enough to warrant such speculation.
Introduction
Fibronectin exists in a soluble protameric form at micromolar concentration in blood plasma and in an insoluble multimeric form in extracellular matrix [ 1,2]. Plasma fibronectin is soluble up to a concentration as high as 20 pM at’physiological pH, ion concentrations and temperature. Unlike fibrillar collagens, laminin, actin and tubulin, therefore, circulating fibronectin does not selfpolymerize in physiologically relevant solutions. There is no evidence for a modifying event, as with the cleavage of fibrinogen to fibrin monomer, to trigger self-polymerization of fibronectin. Finally, there is little passive accumulation of fibronectin in pre-existing extracellular matrix [3,4]. Rather, assembly of plasma tibronectin takes place at specialized areas on the surfaces of cells [5]. A number of cultured cell types, especially fibroblasts, synthesize and secrete forms of libronectin that are spliced differently from the plasma form (see below) and have a greater tendency to self-polymerize [ 21. Assembly of both plasma fibronectin (present in serumcontaining culture medium) and cell-derived fibronectin into extracellular matrix is directed by cultured fibroblasts [3-71. Electron microscopic studies of cultured cells and granulation tissue of healing wounds have demonstrated transmembranous associations of fibronectin-containing extracellular matrix fibers and bundles of actin microftlaments localized at dense submembranous plaques 181. It is likely that insolubilization of fibronectin is part of a complicated process by which the intracellular cytoskeleton determines the pattern of assembly of extracellular matrix and the extracellular matrix determines the pattern of assembly of intracellular cytoskeleton. In the present review, I describe features of fibronectin and the ceU surface that are important in assembly, and propose a working model of the process. There is not a good precedent for the proposed pathway, and much more ceU biology and protein chemistry needs to be done to validate these ideas. The process of assembly 214
Biology
@ Current
Biology
Structure
of fibronectin
Fibronectin is a mosaic protein which comprises a series of repeating modules [1,2]. Mosaic proteins are thought to arise by duplication and shuffling of exons, because the modules are usually encoded by one or two exons, and the intron/exon boundaries have the same phase [9]. The deduced amino acid sequences for libronectins of the clawed frog and rat are well conserved, possessing 71% amino acid identity and the same overall organiTz+ tion [lo*]. Thus, the fibronectin gene must have arisen before the appearance of vertebrates and must then have been constrained in evolution. Figure 1 depicts protameric fibronectin as a dimer of extended subunits. The subunits are similar except for splice variations and contain three types of repeating structural units: 12 type I modules, two type II modules, and 15-17 (depending on splicing) type III modules [ 1,2]. The type I modules are clustered at the aminoand carboxy-terminal regions of the subunits, which are joined by a pair of disulfide bonds at the carboxy-terminal ends. Tandem type II modules are located in the part of fibronectin that binds to gelatin. The type III modules account for -60% of the sequence and are arrayed in the middle. The 15 type III modules that are present in all fibronectin subunits are numbered III-l, III-2, .... III-15 whereas the two type III modules that are subject to alternative splicing are called ED-A (or EIIIA) and ED-B (or EIIIB, where ED stands for ‘extra domain’) [ 1,2]. Such a nomenclature (numbers for modules always present and letters for alternatively spliced modules),avoids the awkward situation, which has been found with tenascin, of having variable numbers of alternatively spliced modules in different species [9]. Between III-14 and III-15 lies the variable (V) or connecting segment (CS) sequence. This Ltd ISSN 0959-440X
Assemblv
of fibronectin
into extracellular
matrix
Mosher
strands of the larger P-sheet was well deIined, whereas the loop between the second and third strands was not highly restrained and apparently has considerable flexibility. Because both the amino- and carboxy-terminal residues of the module lie in P-sheets, there is the possibility that adjacent modulei are linked together through a common o-strand that passes directly from the carboxyterminal P-sheet of one module into the amino-terminal P-sheet of the following module.
sequence is not homologous to other parts of fibronectin and can be completely absent, present in part, or present in full as a result of alternative splicing by exon subdivision [1,2]. The images obtained when fibronectin is sprayed on various non-physiological surfaces are varied j 111. Molecules on mica examined by scanning force microscopy exhibit a V shape oriented parallel to the scan direction [12*]. This result indicates that the arms of the molecules are relatively mobile and that the site for binding to the mica substrate is located near the carboxy-terminal intersubunit disulfides. In contrast, libronectin molecules sprayed on polymethylactylate form a thin network, interpreted as molecules bound to one another [ 12*].
Type II modules
These modules are also found as colinear repeats in bovine seminal plasma proteins PDC-109 and BSPA3, as triple repeats in type IV collagenses, and as single modules in blood coagulation factor XII and insulin-like growth factor receptor II [IS=]. The two-disullide type II modules are homologous to the three-disulhde kringle modules of prothrombin and Iibrinolytic enzymes [l6]. The solution conformation of a proteolytically derived type II module of bovine seminal fluid protein PDC-109 has been determined by proton NMR and molecular dynamics calculations [ 15-l.
Type I modules
Fibronectin type I modules, in addition to appearing 12 times in libronectin, occur once each in blood coagulation factor XII and tissue plasminogen activator. The structure of the seventh type I module of human fibronectin, expressed in yeast (Saccharomyces cere uisiae), was determined in solution using proton NMR spectroscopy [ 131. For comparison, an NMR-derived structure of the type I module of tissue-type plasminogen activator is also now available [14-l.
Two central anti-parallel P-sheets, which lie roughly perpendicular to each other, and two irregular loops support a large, partially exposed, hydrophobic surface which defines a putative binding site (Fig. 2). The disulfide bridges lie approximately perpendicular to each other, as predicted from homology based on the X-ray crystallographic structure of the first kringle domain of bovine prothrombin [ 161. A cluster of aromatic residues are solvent-accessible and form the presumptive exposed hydrophobic surface.
The dominant structural features of the module are two anti-parallel P-sheets, a small double-stranded sheet comprising the amino-terminal third of the sequence and a triple-stranded sheet comprising the carboxy-terminal residues (Fig. 2). Both P-sheets have a right-handed twist and are stacked to enclose a hydrophobic core comprising three highly conserved aromatic residues and the consensus l-3 and 2-4 disuffide bonds. The disulfide bridge between the first and third cysteines links the two P-sheets, and the disuliide bridge between the second and fourth cysteines links two adjacent strands of the larger g-sheet. The turn between the first and second
Type III modules
Over 300 occurrences of type III modules have been found in over 65 proteins, including extracellular ma-
‘We 1 0
Type
II
0
Type
III
Fig. 1. Schematic
C;1\
III~lII~IIIIII q =, v
EDA.
N
6 ED-B
L
2 Gelatin binding
diagram .of the modular structure of a fibronectin dimer. The two subunits are shown with the am’ino termini (N) to the right and the carboxyl termini K) to the left. Regions implicated in matrix assembly (and discussed in the text) are shown on one of the subunits, and sites of alternative splicing and gelatin binding are shown on the other.
215
216
Macromolecular
assemblages Fig. 2. Folding of (a) type I, (b) type II and (c) type Ill modules. The folding of the type III module is shown more schematically so that interactions of the loops that have been demonstrated in different type Ill modules can be shown.
(a)
trix molecules such as iibronectin and tenascin, cytokine receptors and other cell-surface proteins including the receptor for human growth factor, and intracellular proteins involved in muscle filament formation [17e1. TWO structures of type III modules are available: one was solved by multiwavelength anomalous diffraction phasing of the selenomethionyl third type III module of human tenascin expressed in EscberiZbia coli [18**]; the other was determined by NMR of the tenth type III module of human iibronectin expressed in yeast [19**]. The structure of the tenascin type III domain, which has overall dimensions of 40 X 17 x 28% consists of seven j3-strands arranged into two anti-parallel P-sheets, one of four and one of three strands (Fig. 2) [180*]. The folding topology of the libronectin module [lp*] is identical, as are the crystallographically determined topologies of the two extracellular modules of the human growth hormone receptor [ 20*]. Similar folds have also been shown for the D2 domain of CD4 and the bacterial chaperonin PapD. The relevant sequences in CD4 and PapD are not homologous to type III modules, and thus the structures are considered to be related by convergent rather than divergent evolution (18*-J!?*]. The seven ~-strands are labeled A, B, C, C’, E, F and G and form two P-sheets, A-B-E and Cl-C-F-G (Fig. 2). The fold is similar to that of immunoglobulin domains, except that strand C’ is hydrogen-bonded to strand C in the type III module rather than to strand E in the immunoglobulin domain. Both p-sheets have a right-handed twist, and they stack on top of one another to enclose a hydrophobic core.
C
(b)
In the NMR spectra, the lack of long-range interactions for the residues in the loops between strands C and C’ and between strands F and G suggests that these loops are conformationally labile [19**]. The F-G loop is the location of the Arg-Gly-Asp (RGD) sequence which is essential for adhesive activity of the tenth type III module of iibronectin towards the a& integrin receptor [19*-l and of the third type III module of tenascin towards the a$~ integrin receptor [18*-l. Snake venoms contain a number of small, disulfide-rich proteins, the so-called disintegrins, which interact with integrins to block cell adhesion. The RGD motifs of two disintegrins have been shown by NMR to lie at the apices of conformationally flexible loops [21,22]. Thus, two different protein folds can serve as structural frameworks for a flexible, RGDcontaining cell-adhesive loop.
Heparin
C
HCF
Fibronectin ?
Other loops of type III modules have been demonstrated to participate in binding interactions (Fig. 2). In crystals of the human growth hormone-receptor complex, one type III module interacts with the hormone via its A-B and E-F loops, and the second type III module interacts via its F-G and B-C loops [20-l. A major site of heparin binding to fibronectin has been localized by mutagenesis studies to two argininyl residues at the amino terminus of module III-13 [23].
Connectivities
among
modules
Type III modules occur as repeated arrays and, when visualized by electron microscopy, appear as extended and relatively straight rods [ll]. The calculated contribution of each module to the length of the strand is less than the distance between the amino- and carboxy-terminal ends of the isolated module, and the two ends are on the same side of the module [ 18**]. Thus, successive modules must be tilted relative to one another, and it is likely that alternate modules are rotated by 180”. Such an arrangement might serve to reduce the flexibility of type III arrays. Although the modules are independently folded units [24=], arrays of modules interact to form larger structural and functional domains. The adhesive activity of the RGD-containing tenth type III module of fibronectin is enhanced if the tenth module is in an array that includes III-8 and III-9 1251. A monoclonal antibody specific for fibronectin containing the variably expressed ED-B type III module recognizes an epitope in III-7 which is cryptic in an array of III-7, III-8, and III-9 but unmasked when ED-B is inserted between III-7 and III8 [26*]. Conversely, a monoclonal antibody specific for libronectin that lacks ED-B recognizes III-7 and III-8 in an array lacking the inserted ED-B [ 26.1. Recognition of fibronectin by a monoclonal antibody that blocks assembly (see below) requires that the last type I module of the gelatin-binding region (I-9) be contiguous to the first type III module (III-l) and is sensitive to the reduction of disuliides in I-9 [27]. Finally, binding of the amino-terminal region of iibronectin to cellular assembly sites (see below) requires that the first five type I modules (I-l to I-5) be in an array, such that deletion of any one unit results in the loss of binding activity [28].
Features
of fibronectin
required
Assemblv
of fibronectin
Interchain
disulfide
into extracellular
matrix
Mosher
bridge
The deletion of 20 amino acids from the carboxyl terminus of Iibronectin, including the cysteines involved in interchain disuffide bridges, yields monomeric molecules that do not become incorporated in matrix, i.e. only molecules containing the. bridge region are assembled [31]. Structures of the proteolytically derived heptapeptides, (Val-Gln-Cys-Pro-Ile-Glu-Cys)z, which include the bridge region have been determined by NMR in both water and dimethylsulfoxide [32*-l. The structures provide strong evidence that, although the two subunits are chemically linked head-to-tail in an antiparallel fashion (Fig. l), relative to each other the two chains are effectively disposed in a parallel fashion. Thus, the carboxyterminal bridges act as a terminal which holds the two chains together rather than as a linear junction between mutually extending chains. In addition, the cyclic peptide, which is constrained by the two cystine bridges and the proline residues, is conformationally labile, as shown by substantially different conformations in water and dimethylsulfoxide [ 32**].
Amino-terminal
domain
Most of the binding activity that directs fibronectin to as-‘ sembly sites is contained in the amino-terminal 70 kDa region of the fibronectin subunit, in particular within modules I-l to I-5. Although proteolytic fragments or recombinant proteins containing these modules do not assemble into Iibrils, these proteins bind to assembly sites with the same avidity as intact fibronectin and block the binding and assembly of intact fibronectin [27-291. Removal of any of modules I-l to I-5 results in a protein with an ai%niry for assembly sites 7-lOO-fold less than the nonmutant protein [28]. Mutation of a conserved tyrosine in any one of the modules also causes loss of binding affiity, although the decreases are not as pronounced as in the deletion set. Similar results have been obtained for dimeric fibronectin constructs; molecules containing all five amino-terminal type I modules are incorporated into matrix, whereas molecules lacking module I-5 are not [31].
for assemblv
Fibronectin matrix assembly is a multistep process that requires the presence of viable cells [3,4,29,30]. In the first step, fibronectin binds to the cell surface (Fig. 3). This binding is saturable and reversible. In the following steps, interactions between or among fibronectin molecules lead to the formation of insoluble libronectin multimers. In a presumptive final step, the binding site is regenerated. Three kinds of experiments have been done to probe the features of fibronectin that are required for assembly. First, proteolytic fragments of plasma iibronectin have been tested for their ability to bind to assembly sites and interfere with the binding and assembly of Intact fibronectin. Second, monoclonal antibodies to Iibronectin have been tested for their ability to block binding and assembly. Third, altered iibronectins generated by recombinant techniques have been tested for their ability to assembleinto matrix.
The most likely explanation for the requirement of all five amino-terminal type I modujes for binding activity is that the live modules form a single functional unit, so that deletion of one module or disruption of its structure by mutation of a conserved hydrophobic residue disrupts the structure and function of the entire unit. The ftve modules are also thought to function as a unit in the phenomenon of ‘matrix-driven translocation’ [33=]. This is a phenomenon in which interactions between. distinct regions of an artificial extracellular matrix cause the rapid, unidirectional transport of suspended cells or latex particles into previously unpopulated regions of the matrix.
Presumptive
self-interaction
site
Another functional unit of iibronectln which is probably important in assembly comprises modules I-9 and
217
218
Macromolecular
assemblages
I
I
. I
I
-
~
I Muitimer Membrane
I
I
_ I
I
_
^
I Fig. 3. Model of the proposed cycle of assembly. The protamer, arranged as in Fig. 1, binds in an extended conformation to a cell-surface binding site that includes a hypothetical binding molecule (wedge), CL& integrin (paired vertical bars), and already assembled fibronectin molecules. The change from reversibly bound to irreversibly bound protamer is hypothesized to occur in the intersubunit disulfide bridge region, as represented by the open diamond. Localization and regeneration of assembly sites is controlled by intracellular cytoskeletal elements and associated kinases tarrows).
III-1 [ 271. Two monoclonal antibodies to this region in-
hibit assembly of fibronectin. One antibody recognizes a conformationalIy sensitive epitope which requires intact disulfides io I-9 and contiguity of I-9 with III-l. The second antibody recognizes a linear epitope in LI-1, probably in the E-F loop ([27], also see [19-l >. A gelatin-binding fragment of fibronectin containing this functional unit inhibits the binding of fibronectin to cell layers IO-fold better than does a fragment lacking LLI-1 and the two epitopes. The fragment containing III-1 and the two epitopes does not bind to cell layers, suggesting that inhibition results from binding of the fragment to protameric fibronectin rather than to the assembly sites 1271. A peptide based on a sequence in N-1, however, does bind to cell layers and to intact fibronectin, and might represent a cryptic binding
site that becomes functional during matrix assembly and mediates libronectin-fibronectin interactions [34-l. Two observations indicate that the I-g/III-l sequences might not be crucial for assembly. First, III-1 is one of the less conserved regions of fibronectin [lo]. Second, dimeric fibronectin constructs lacking III-1 or I-9 and III-1 do assemble into matrix [ 31,35**].
Cell
adhesion
site
Yet another feature of fibronectin which is probably important for assembly is the region recognized by the cr5p1 cell-adhesion receptor. This region encompasses III-B, IlI-9 and III-lo, the latter containing the RGD-containing F-G loop. Monoclonal antibodies to this region of
Assembly
libronectin inhibit elaboration of Iibronectin-containing fibrils by cultured fibroblasts [36]. Also, monoclonal antibodies to a5pI block assembly, as described below. Yet, surprisingly, deletion of the RGD sequence for dimeric fibronectin constructs does not interfere with their incorporation into matrix [31].
Carboxy-terminal
modules
When the carboxy-terminal type 1 module regions are fused as a recombinant protein with the amino-terminal modules, the resulting dimeric protein is highly capable of assembling into matrix [35=*]. The carboxy-terminal type I modules, even when joined by the carboxy-terminal disuffide bridges, do not become incorporated into matrix [37]. Mutation or deletion of the carboxy-terminal type I modules in full-length iibronectin does not nevertheless impair grossly the abilities of the altered proteins to be assembled into iibrils [38**]. It is likely, therefore, that the carboxy-terminal type I modules are not involved signiiicantly in assembly.
Features of the cell surface assembly
required
for
The molecules at assembly sites that mediate binding of the amino-terminal modules of fibronectin are not known. Nothing from detergent extracts of iibroblasts competent in assembly binds to an affinity column containing the amino-terminal fragment [39]. Monoclonal antibodies to the as& integrin inhibit binding and assembly of iibronectin by cells [39,40], but also inhibit binding of the amino-terminal ilbronectin fragment, even though there is no evidence that the integrin binds to the fragment [39]. The inhibitory effect of the antibody is evident only after a 10-15 min incubation period. These results suggest that integrins are required for expression of the binding sites for the amino-terminal modules of fibronectin but do not interact with the modules per se. Consistent with this idea, over-expression of a results in increased deposition of Iibronectin matrix r411. Cross-linking studies with transglutaminase and chemical cross-linkers have also been performed [42,43]. Both approaches demonstrate prominent cross-linking of bound amino-terminal fragment to intact iibronectin. Chemical cross-linking studies also identify an - 120 kDa protein which is not aspI [43]. Summing up, then, the only molecule known for sure to interact with the aminoterminal region of fibronectin is fibronectin itself. A major problem in the identification of cell-surface molecules important for fibronectin assembly is that the assembly sites are so labile. For example, transforming growth factor-8 causes a rapid gain of assembly sites [441, whereas agents that elevate intracellular CAMP concentration cause rapid loss of assembly sites [45]. Some cell lines require furthermore the presence of serum for efficient matrix formation [8,46-l. The enhancing activity is enriched ln lipoprotein fractions of serum [46*] and
of fibronectin
into extracellular
matrix
Mosher
can be duplicated by low concentrations of lysophosphatidic acid (WJ Checovich, DR Mosher, unpublished data). Cytochalasin [4] and inhibitors of protein kinase C (CE Somers, DF Mosher, unpublished data) cause prompt ( < 5 min in the case of the protein kinase C inhibitors) loss of cell-surface binding sites for fibronectin or its amino-terminal fragments. The effect of the protein kinase C inhibitors is rapidly reversed upon their removal (CE Somers, DF Mosher, unpublished data). These agents which perturb the binding and assembly of’ libronectin also have profound effects of actin-containing stress fibers and the ability of cells to grip the substratum through focal contacts. Lysophosphatidic acid or serum rapidly stimulates stress fiber and focal adhesion formation in serum-starved iibroblasts [47**]. Conversely, inhibitors of protein kinase C reduce stress fiber and focal adhesion formation [48*-l. The determinants of focal adhesion and stress fiber formation are many, including the GTE-binding protein rho [470*], protein kinase C [48==], tyrosine kinases including focal adhesion-associated kinase [49-511, kinase substrates such as paxillin [49], and, of course, integrins. Integrin-mediated adhesive contacts of motile libroblasts on a substratum demonstrate limited fluctuation in size, density and shape [52*-l. The rapidity with which assembly sites are up- and down-regulated suggests that the same machinery is used in a much more dynamic fashion in matrix assembly. Fibroblasts cultured in serum-containing medium can be considered as the equivalent of specialized fibroblast-like cells, called myofibroblasts, which are present in tissues undergoing contraction [53*]. Tissue contraction is a part of normal wound healing. Cultured libroblasts generate tension, and it is the tension-generating cells that assemble fibronectin matrix [53-l. The association between tension and elaboration of matrix is of potential importance clinically. For instance, stretching of glomerular mesangial cells by glomerular hypertension provokes increased production of extracellular matrix molecules i541.
Model
for assembly
Figure 3 presents a model for assembly that tries to account for the observations described above. The assembly site is shown as being controlled by cytoskeletal and focal adhesion molecules linked to & kitegrins. Fibronectin binds reversibly to the site via its amino-terminal type 1 modules. The fibronectin binds to a yet-to-beidentified molecule, to an altered part of already bound iibronectin, or to an altered integrin. . Something then happens so that fibronectin becomes bound irreversibly as part of a large multimer. The multimer resists dissociation by sodium dodecyl sulfate unless a reducing agent is also present. Such behavior can be considered as evidence for a thiol-disuliide exchange resulting in the formation of disuliides among protamers [29,30]. My laboratory has made a considerable effort to identify the newly formed disulfides and had no success
219
220
Macromolecdar
assemblages
at all. Thus, I presently favor the hypothesis that there is a conformatio@ change of polymerizing molecules, leading to protein-protein interactions that resist dissociatibn. The changed conformation is hypothesized to be stabilized by disulfides. A prime candidate for the conformationally labile region is the carboxy-terminal dlsutide bridge (shown as a change from the open circle to the open diamond in Fig. 3). In a presumptive final step, the binding site is regenerated. This is shown as the cytoskeletal array pulling the integrin and amino-terminal binder in the plane of the membrane, thus focusing the assembly machinery on the untethered end of the newly insolubilized protamer. This recycling is presumably the step that is interrupted by protein kinase C inhibitors or anti-integrin antibodies. The model shown in Fig. 3 applies to addition of a libronectin protamer at the end of a growing fibril. There must be a condensation phase to initiate fiber formation, perhaps an alignment of integrins to bring fibronectin molecules in appostion. There must also be fixation of the non-growing end of the fiber so that the fiber can be tensioned.
Concluding
comments
and future
directions
Our current understanding of the fibronectin assembly process is hardly satisfactory, despite the efforts of a number of laboratories. The only parts of libronectin that may be needed for assembly are the 6ve amino-terminal type I modules and the carboxy-terminal disulfide bridges. How would one then explain the evidence for a critical role of aspI? And how would the cell keep track of the growing end of the fibril? None of the published experiments of matrix assembly in cell culture have been performed without the confounding presence of endogenous fibronectin, either intact or as a heterodimer with recombinant protein. The generation of cell lines that provide just such a null background is important to progress, as is the production of informative recombinant proteins. More studies such as the recent demonstration of a periodic distribution of the ED-A module within a fibril [%I are needed to learn whether or not fibronectin is @deed extended within a fibril and, if so, with what overlap. Finally, the long-standing questions of what b&is the amino-terminal modules to assembly sites and what forces are responsible for holding the multimer together need to be answered.
Acknowledgements
Supportedby grants from the NationalInstitutesof Health and the Wisconsin Affiliate of the American Heart Association.
References
and recommended
reading
Papersof particularinterest, published within the annual period of review, have been highlighted as: of special interest . . .. of outstanding interest 1.
PETERSENTE, SKORSTENGAAIUJ K, VIBE-PEDERSENK: Primary Structure of Fiironectin. In Fibronecfin. Edited by Mosher 0. San Diego: Academic Press; 19891-24.
2.
HYNESRO: Fibronectins
3.
CUR WG: The Role of Intermolecular Disulfide Bonding in Deposition of GP140 in the Extracellular Matrix. / Ccl/ Biol 1984, 99:105-114.
4.
BARRY ELR, MOSHERDF: Factor XIII Cross-Linking of Fibronectin at Cellular Matrix Assembly Sites. J Viol u3etn 1988, 263:10464-10469.
5.
PETERSDP, MOSH~?ROF: Localization of Cell Surface Sites Involved in Fibronectin Fibrillogenesis. / Cell Biol 1987, 104:121-130.
6.
ALUO ‘AE, McKEow?+LONGO PJ: Extracellular Matrix Assembly of Cell-Derived and Plasma-Derived Fibronectins by Substrate-Attached Fibroblasts. J Cell P.!ysiol 1988, 135:459-466.
7.
P~IIS DMP, PORTZ IA, FUUENWIDER J, MOSHEH DF: CoAssembly of Plasma and CeUular Fibronectins into FibrUs in Human Fibroblast Cultures. J Ceil Biol 1990, 111:249-256.
8.
SINGER II: Fibronectin-Cytoskeleton Relationships. In Fibronech Edited by Mosher 0. San Diego: Academic Press; 1989:13~161.
9.
PATCHYL: Modular Exchange Principles in Proteins. CLOT Opin SWuc~ Biol 1991, 1:351-361.
New
York: Springer-Vedag; 1990.
10. .
D~SIMONE DW, NORTON PA, HYNES RO: Identification and Characterization of Alternatively Spliced Fibronectin mRNAs Expressed in Early Xenopus Embryos. Dee Biol 1992, 1491357-369. There are now a number of species for which the sequence of Iibronectin is known, including human, mt, cow, and Xenopus, and the comparisons may give important perspectives on features of fibronectln that are critical for its function. 11.
ODIXMATT E, ENCELJ: Physical Properties of Fibronectin. In Fibronecrh Edited by Mosher OF. San Diego: Academic Press; 1989:2545.
EMCH R, ZENHAUSERN F, JOBIN M, TABORELLIM, DESCOIJIXP: Morphological Difference between Fibronectin Sprayed on Mica and on PMMA UIfrumicmsco~ 1992, 4244:1155-1160. This paper, which presents images of fibronectin sprayed on surfaces and studied by scanning force microscopy in air, complements previ. ous rotary shadowing and scanning transmission electron microscopic sNdies [ll]. 12.
.
13.
BARONM, NORMAN0, WILLIS4 C,~PL)ELLID: Structure of the Fibronectin Type 1 Module. Nalure 1990, 345642-646.
14. .
DOWNINGAK, Druscou PC, HARVEYTS, DUDGEONTH, SMITH BO, BARONM, CAMPBEUID: Solution Structure of the Fibrin Binding Finger Domain of Tissue-Me Plasminogen Activator Determined by ‘H Nuclear Magnetic Resonance. / /MO/ Biol 1992, 225:821-833. The overall fold shows a striking similarity to that of module 1-7 of fibronectin [13]. One significant difference between the two molecules is that hydrophobic residues cover the exposed surface of the major P-sheet region. It is suggested chat one face of this region interacts with anoiher part of the tissue-type plasminogen activator. 15. .
CONSTANI-INE
KL,
MADRID
M,
BANW
I
TTIEXER
M,
PATIHY
I, LUNDM: Refined solution Structure and Ligand-Binding
Properties of PDC-109 Domain B: a Collagen-Binding II Domain.J Mol Biol 1992, 223:281-298.
Type
Assembly
of fibronectin
into extracellular
matrix
221
Mosher
The authors present a structure which is more refined than that reported by the same authors previously and which is more like the structure predicted in [ 16). A hydrophobic binding pocket in the structure is perturbed specifically by leucine analogs, suggesting that this pocket is used to recognize leucine. and/or isoleucine-containing sequences in collagen. HOLLAND SK, HAWX K, BLWE CCF: Deriving the Genedc 16. Structure of the Fibronectin Type II Domain from the Prothrombin Kringle I Crystal Structure. EMEO / 1987, 6:1875-1880.
26.
17. .
27.
CHERNOUSOVMA, FOGER’IY FJ, KOTE~ANSKY VE, MOSHER DF: Role of the I-9 and III-1 Modules of Fibronectin In Formation of an Extracellular Fibronectin Matrix. J Biol Ckm 1991, 266:10851-10858.
28.
Sorrw
BOW P, IXQUITLE RF: Proposed Acquisition of an Animal Protein Domain by Bacteria. Proc Null Acad U 5 A 1992, 8989908994. A systematic screen of a protein database turns up over 300 sequences that meet the criteria for a iibronectin type III module in a wide range of proteins, including bacterial carbohydrate-splitting enzymes. Because type III modules are not found in any fungal or plant proteins, it is suggested that the bacterial modules were acquired by horizontal gene transfer from an animal source. This paper provides strong arguments for this sutprisi.ng conclusion. IEWY DJ, HENDRICKSON WA AUKHIL I, ERICKSON HP: Structure l . of a Fibronectin Type III Domain from Tenascin Phased by MAD Analysis of the Selenomethionyl Protein. Science 1992, 258~987-991. The production of a selenomethionine-substituted protein enables the anomalous diffraction signal from Se to be used to solve the crystallographic phase problem. The structure is refined to 1.8.& resolution. Color graphics, especially of a space-iilling model of a possible alignment type 111modules in a repeated army, make reading this article very worthwhile. Complements the dynamic information in [ I9**]. MAIN
AI,
HARWY
TS,
BARON
M, BOYD
J, C~~IPBELL
ID:
The
Three-DimensionaI Structure of the Tenth Type III Module of Fibronectin: an Insight into RGD-Mediated Interactions. Cell 1992, 71:671-678. A more refined structure than that reported by the same authors previously. An analysis based on 36 final structures, calculated using sim. ulated annealing protocols, is presented. This study complements the static information in [ 18**]. The flexible, dynamic nature of two of the loops when viewed in stereo is particularly impressive. 20. DE Vos AM, ULTSCH M, KO%XXOF~ AA: Human Growth Hor. mone and Extracelhtlar Domain of Its Receptor: Crystal Structure of the Complex. Science 1992, 255:306312. This study gives possible insight into protein-protein interactions that involve more than one type III module. The extracellular domain of the receptor contains two type III modules. In the complex, one hormone molecule interacts with two receptor molecules, and all four modules in the complex contribute residues that participate in hormone binding. 21. ALXER MA, LVARUS RA, DENNIS MS, WAGNER G: Solution Structure of Kistrin, a Potent Platelet Aggregation Inhibitor and GPIIb-IIIa Antagonist. Science 1991, 253445-448. 22. SAUDEKV, ATKINSONRA, PELTONJT: Three-Dimensional Stntcture of Echistatln, the Smallest Active RGD Protein. Bio dwmr3t?y 1991, 30:736+7372. ..
BARKALOWFJB, SZHWARZBALIER JE: Localization of the Major Heparin-Binding Site in Fibronectin. 1 Biol Chem 1991, 266:7812-7818. 24. ~NOVICH SV, Novowxwy VV, BREW SA, INGHAM KC: Re. versible Unfolding of an Isolated Hapatin and DNA Binding Fragment, the First Type III Module from Fiironectin. Biocbim Biophvs Acra 1992, 111957-62. Several fragments containing all or part of the first type III module are isolated, and their folding properties are studied by fluorescence spec troscopy and dlfferentiaI scanning caIorimetty. The results indicate that the module can fold independently of neighboring modules and is corn. posed of a stable core flanked by less compact regions. 25. AOTA S, NAW T, YAMADAKM: Characterization of Regions of FIbronectIn besides the ArglnIne-Glycine-Aspartic Acid Sequence Required for Adhesive Function of the Cell-Binding Domain Using Site-DIIected Mutagenesis. / Biol &em 1991, 266:15938-15943. 23.
B,
IEPflINt
A,
&MANN1
G,
SAGltUTi
M,
ZUtDt
L The Inclusion of the Type III Repeat ED-B In the Flbronectin Molecule Generates Conformational ModiEcations that Unmask. a Cryptic Sequence. / Biol C4em 1992, 267~24689-24692. ED-B is highly conserved (100% identity between rat and human) and should not be antlgenlc. This paper solves the mystety of how it is possible to produce an antibody that recognizes fibronectin with ED-B, and calls attention to the importance of the inter-modutar interactions to structure and function.
J, SXWARZBAUER
J, SEUZGUE J, MOSHER DF:
Five
Type
I Modules of Fibronectin Form a Functional Unit that Binds to Fibroblasts and Staphylococcus aweus J Biol C&em 1991, 266:12840-12843. 29.
18.
19.
CARt+EhiOUA
.
MCKEOWN-LONGO
PJ, MOSHER
DF:
The
Assembly
of
tbe
Fi-
bronectin Matrix in Cultured Human Fibroblast Cells. In Fibronecfin. Edited byMosher DF. San Diego: Academic Press; 1989163-179. Mechanisms for Organization of FiMatrix. Cell D#erenliution Deu 1990, 32:439-450.
30.
FOGERTY
31.
S~HWARZBAUER
bronectin
FJ, MOSHER DF:
JE:
Identihcation
of
the
FibronectIn
Se-
quences Required for Assembly of a FibrIIIar Matrix. / Cell Biol 1991, 113:1463-1473.
32. ..
AN SSA, JIMBNEZ-BARBERO J, PETERSEN TE, LUN& M: The Two Polypeptide Chains in Fibronectin Are Joined in AntiparalIel Fashion: NMR Structural Characterization. Biocbemt3ry 1992, -319927-9933. Structures ot me proteoIytIcaIIy derived carboxy-terminal disulhde
bridge region of libronectin are determined in both water and dimethylsulfoxide. The surprising finding that the two subunits are chemically linked head-to-tail in an antiparallel fashion but are structurally parallel might reconcile the apparent discrepancy between electron microscopic evidence for a V-shaped molecule and chemical evidence for an antiparallel array. 33. .
JAW&IA NS, ROSENFEU)L, KHAN MY, DANWEFSW I, NEV&WN SA: Interaction of Fibronectin with Hepatin ln Model Extracelhtlar Matrices: Role of ArgInine Residues and Sulfate Groups. Biocbemr3~y 1991, 30:15381544. This is the last in a series of papers describing ‘matrix-driven transloca~ tion’. The relationship of this phenomenon to matrix assembly Is not proven, but the structure-function correlations are very similar. MOR~AA, RUOS~AH~E: A Fibronectm Self-Assembly Site Invalved in Fibronectin Matrix Assembly: Reconstruction in a Synthetic Peptide. J Cell Eiol 1992, 118:421429. A successful search for a peptide based on the sequence of III-1 that will bind to Iibronectin. The active sequence includes the B-C loop, strand C, the CC’ loop, and strand C’ pig. 2).
34. .
35.
ICHIHARA-TANAKA
K, MAEDA T, TITANI
K, SEKIGUCHI
K
Mat&t
Assembly of Recombinant FibronectIn Polypeptide Consisting of Amino-Terminal 70 kDa and Carboxyl-Terminal 37kD Regions. FEBS Lett 1992, 299:15>158. This study compares metabolism by L cells of recombinant proteins containing each of the two regions alone with a protein contaktktg the two regions Fused together. The fused form is found in extracellular mauix. A major problem with the use of such an approach in thII paper and in [ 31 I is the formation of heterorkmers of intact endogenous iibronectin subunits and the recombinant protein. Thus, it is dklicult to know whether the recombinant protein becomes incotporated Into matrix by virtue of its own attributes or by virtue of the endogenous subunit to which it is linked. ..
36.
MCDONALDJA, QUADE BJ, BROEKE~MANN TJ, IACHANCE R, FORshw K, HASEGAWAE, AKWAMAS: Fibronectbt’s Cell Adhesive Domain and an Amino-Terminal Mattix Assembly Domain Pat-bcipate In Its Assembly In FIIrobIast PeticeUular Matrix. J Biol c%m 1987, 262:2957-2967.
222
Macromolecular 37.
assemblages
ICHIHARA-TANAKA K, TITANI K, Carboxyl-Terminal Fibrin-Binding bronectin Expressed in Mouse 265401407.
SEKICUCHI K: Recombinant Domain of Human FiL Cells. J Biol Umn 1990,
Son;ll~ J, MOSHER DF: Assembly of Fibronectin Molecules with Mutations or Deletions of the CarboxylsTerminal Type I Modules. BicchentLW-j~ 1993. in press. Non-mutant, mutated and deleted rat fibronectins are produced in monkey COS cells, immunodepleted of monkey libronectin. and studied in CUlNreS of human fibroblasts treated with qcloheximide in order to block the synthesis of human fibronectin. Alterations ofi the carboxy-terminal type I modules have little or no effect on assembly. This SNd, exemplifies the tenuous arguments that can be made follcvwing this type of approach, and the critical need for cells that lack endogenous fibronectin, so enabling the secretion and assembly of recombinant proteins to be studied against a null background.
38. ..
Stress Fibers in Response to Growth 70~389-399. Although lysophosphatidic acid is not mentioned found effects of this lysophospholipid on focal stress fibers form a major part of this paper.
BURIUDC~ K, TURNER CE, ROSIER LH: Tyrosine of Paxillin and pp125Fm Accompanies Extracellular Matrixz a Role in Cytoskeletal Biol 1992. 119:893-903.
50.
GI.IA,Y J-L. SHAUO~AY D: Regulation of Focal AdhesionAssociated Protein Tyrosine Kinase by Both Cellular Adhesion and Oncogenic Transformation. Nnlrrre 1992, 358:690-692. KORI\~)ERG I+ EARP HS. PAKSONS JT. SCHAILLR M, JUUANO RL Cell Adhesion or lntegrin Clustering Increases Phosphoryfation of a Focal Adhesion-Associated Tyrosine Kinase. J
40.
AKNAMA SK, Fibronectin ies: Roles in Cytoskeletal
YAMADA SS, CMN WT. YAMADA KM: Analysis of Receptor Function with Monoclonal AntibodCeU Adhesion, Migration, Matrix Assembly and Organization. J Cd BioI 1989, 109:863-875.
51.
41.
GL%YCOIII F, RUOSI~I E: Elevated Levels of the a$, Fibronectin Suppress the Transformed Phenotype of Chinese Hamster Ovary Cells. Cell 1990, 60849-859.
52. ..
42.
BARRY ELR, MOSHER DF: Factor XIIla-Mediated Cross-Linking of Fiironectin to Fibroblast Cell Layers: Cross-Linking of Cellular and Plasma Fibronectin and of Amino-Terminal Fibroneqin Fragments. J Rio/ Uxm 1989, 264:417+il85.
43.
LIMPER AH, QUADE BJ, RANGWAIA TS, MCDONAD Bind Fibronectin’s Matrix 1991, 266:9697-9702.
UJC~I
in the title, the proadhesions and actin
49.
FOGERN FJ, AKIYA,\IA SK, YAMADA KM, MOSHER DF: Inhibition of Binding of Fibronectin to Matrix Assembly Sites by Antilntegrin (a$,) Antibodies. J Cc/I BioI 1990, 111:699-708.
TM, that
Cc/I 1992,
48. WOODS A, COUCHMAN JR: Protein Kinase C IfWOkment in .. Focal Adhesion Formation. J Cell Sci 1992, 101:277-290. Inhibitors of protein kinase C are shown to inhibit strongly the formation of focal adhesions and stress fibers in cells spreading on a fibroncytin-coated substratum. Interestingly, P,-integrin subunits do not disperse from the focal adhesions in response to the inhibitors.
39.
L&HXVCE RM, BIRDENMEIER JA: CeU Surface Molecules Assembly Domain. J BioI
Factors.
Biol dent
Phosphorylation Cell Adhesion to Assembly. J Cd/
1992, 267~23439-23442.
REGEN CM, I-lOR\Y117. AF: Dynamics of p, Integrin-Mediated Adhesive Contacts in Motile Fibroblasts. J Cc/I Rio/ 1992, 119:1347-1359. Detailed time-lapse and immunofluorescence studies of the distribution of PI in motile cells. This sNdy provides important data on the amount of integrin that is left on the substratum versus the amount that is apparently recycled and on the rates of the two processes.
TO~WEK JJ, HAAKS&IA CJ, EDDY RJ. VALIG~~ MB: Fibroblast Contraction Occurs on Release of Tension in Attached Collagen Lattices: Dependency on an Organized Cytoskeleton and Serum. Am! Ret 1992, 232:359-368. An informative model of libroblasts CUlNrd in collagen lattices. When the lattices are attached to the dish, extensive arrangements of colinear actin stress fibers occur and surface-associated tibronectin librils are fomied. These disappear when the lattices are detached from the dish and allowed to contract. 53. .
44.
AUEN.HOFFMANN BL, CRANKSHAW CL, MOSHER DF: Transforming Growth Factor Beta Increases CeU Surface Binding and Assembly of Exogenous (Plasma) Fibronectin by Normal Human Fibroblasts. MO/ CeII Biol 1988, 8:423-242.
45.
AUEN-HOFFMANN BL MOSHER DF: Matrix Assembly Sites for Exogenous Fibronectin Are Decreased on Human Fibroblasts after Treatment with Agent which Increase Intracellular CAMP. J Biol Cbetn 1987, 262:14361-14365.
54.
RISER BL, CORI-ES P. ZHAO X, BERN~IN J. DLILI~R F, NARINS RG: lntraglomerular Pressure and Mcsangial Stretching Stimulate ExtraceUular Matrix Formation in the Rat. ./ C/in ftz~~est 1992, 90:1932-1943.
CHECOV~CH YQ, SCHULI-L RL, MOSHER DF: Lipoproteins Enhance Fibronectin Binding to Adherent CeUs. A,-/e,-iad 7lmmbas 1992, 12:1122-1130. The authors describe several lipoprotein fractions that increase the number of binding sites for libronectin on several different cell [ypes.
55.
D%t\hlBA bronectin Cultures.
46. .
47. ..
RIDLEY AJ, ti Regulates the
A- The Assembly
Small GTP-Binding of Focal Adhesions
Protein and
rho Actin
BJ, Pt%aS DMP: Arrangement of Cellular Fiin Noncollagenous FibrUs in Human Fibroblast J Cdl Sci 1991, 100:605-612.
DF Mosher, Departments University of Wisconsin, 53706, USA
of Medicine 1300 University
and Biomolecular Avenue, Madison,
Chemistry, Wisconsin
into extracellular
matrix
Deane F. Mosher University
of Wisconsin,
Madison,
USA
Assembly of fibronectin into a fibrillar matrix is part of a complicated process by which the intracelltilar cytoskeleton determines the pattern of assembly of extracellular matrix and vice versa. Assembly is restricted to dimeric fibronectin molecules with intact amino-terminal modules and occurs at specialized cell-surface assembly sites. Cellular display of assembly sites is labile and requires that cells be under tension and have functional & integrins. Assembly is described as a stepwise event in which protameric dimers bind to assembly sites, multimerization occurs, and assembly sites are regenerated. Current
Opinion
in Structural
1993, 3:214-222
is however of considerable biological importance and interesting enough to warrant such speculation.
Introduction
Fibronectin exists in a soluble protameric form at micromolar concentration in blood plasma and in an insoluble multimeric form in extracellular matrix [ 1,2]. Plasma fibronectin is soluble up to a concentration as high as 20 pM at’physiological pH, ion concentrations and temperature. Unlike fibrillar collagens, laminin, actin and tubulin, therefore, circulating fibronectin does not selfpolymerize in physiologically relevant solutions. There is no evidence for a modifying event, as with the cleavage of fibrinogen to fibrin monomer, to trigger self-polymerization of fibronectin. Finally, there is little passive accumulation of fibronectin in pre-existing extracellular matrix [3,4]. Rather, assembly of plasma tibronectin takes place at specialized areas on the surfaces of cells [5]. A number of cultured cell types, especially fibroblasts, synthesize and secrete forms of libronectin that are spliced differently from the plasma form (see below) and have a greater tendency to self-polymerize [ 21. Assembly of both plasma fibronectin (present in serumcontaining culture medium) and cell-derived fibronectin into extracellular matrix is directed by cultured fibroblasts [3-71. Electron microscopic studies of cultured cells and granulation tissue of healing wounds have demonstrated transmembranous associations of fibronectin-containing extracellular matrix fibers and bundles of actin microftlaments localized at dense submembranous plaques 181. It is likely that insolubilization of fibronectin is part of a complicated process by which the intracellular cytoskeleton determines the pattern of assembly of extracellular matrix and the extracellular matrix determines the pattern of assembly of intracellular cytoskeleton. In the present review, I describe features of fibronectin and the ceU surface that are important in assembly, and propose a working model of the process. There is not a good precedent for the proposed pathway, and much more ceU biology and protein chemistry needs to be done to validate these ideas. The process of assembly 214
Biology
@ Current
Biology
Structure
of fibronectin
Fibronectin is a mosaic protein which comprises a series of repeating modules [1,2]. Mosaic proteins are thought to arise by duplication and shuffling of exons, because the modules are usually encoded by one or two exons, and the intron/exon boundaries have the same phase [9]. The deduced amino acid sequences for libronectins of the clawed frog and rat are well conserved, possessing 71% amino acid identity and the same overall organiTz+ tion [lo*]. Thus, the fibronectin gene must have arisen before the appearance of vertebrates and must then have been constrained in evolution. Figure 1 depicts protameric fibronectin as a dimer of extended subunits. The subunits are similar except for splice variations and contain three types of repeating structural units: 12 type I modules, two type II modules, and 15-17 (depending on splicing) type III modules [ 1,2]. The type I modules are clustered at the aminoand carboxy-terminal regions of the subunits, which are joined by a pair of disulfide bonds at the carboxy-terminal ends. Tandem type II modules are located in the part of fibronectin that binds to gelatin. The type III modules account for -60% of the sequence and are arrayed in the middle. The 15 type III modules that are present in all fibronectin subunits are numbered III-l, III-2, .... III-15 whereas the two type III modules that are subject to alternative splicing are called ED-A (or EIIIA) and ED-B (or EIIIB, where ED stands for ‘extra domain’) [ 1,2]. Such a nomenclature (numbers for modules always present and letters for alternatively spliced modules),avoids the awkward situation, which has been found with tenascin, of having variable numbers of alternatively spliced modules in different species [9]. Between III-14 and III-15 lies the variable (V) or connecting segment (CS) sequence. This Ltd ISSN 0959-440X
Assemblv
of fibronectin
into extracellular
matrix
Mosher
strands of the larger P-sheet was well deIined, whereas the loop between the second and third strands was not highly restrained and apparently has considerable flexibility. Because both the amino- and carboxy-terminal residues of the module lie in P-sheets, there is the possibility that adjacent modulei are linked together through a common o-strand that passes directly from the carboxyterminal P-sheet of one module into the amino-terminal P-sheet of the following module.
sequence is not homologous to other parts of fibronectin and can be completely absent, present in part, or present in full as a result of alternative splicing by exon subdivision [1,2]. The images obtained when fibronectin is sprayed on various non-physiological surfaces are varied j 111. Molecules on mica examined by scanning force microscopy exhibit a V shape oriented parallel to the scan direction [12*]. This result indicates that the arms of the molecules are relatively mobile and that the site for binding to the mica substrate is located near the carboxy-terminal intersubunit disulfides. In contrast, libronectin molecules sprayed on polymethylactylate form a thin network, interpreted as molecules bound to one another [ 12*].
Type II modules
These modules are also found as colinear repeats in bovine seminal plasma proteins PDC-109 and BSPA3, as triple repeats in type IV collagenses, and as single modules in blood coagulation factor XII and insulin-like growth factor receptor II [IS=]. The two-disullide type II modules are homologous to the three-disulhde kringle modules of prothrombin and Iibrinolytic enzymes [l6]. The solution conformation of a proteolytically derived type II module of bovine seminal fluid protein PDC-109 has been determined by proton NMR and molecular dynamics calculations [ 15-l.
Type I modules
Fibronectin type I modules, in addition to appearing 12 times in libronectin, occur once each in blood coagulation factor XII and tissue plasminogen activator. The structure of the seventh type I module of human fibronectin, expressed in yeast (Saccharomyces cere uisiae), was determined in solution using proton NMR spectroscopy [ 131. For comparison, an NMR-derived structure of the type I module of tissue-type plasminogen activator is also now available [14-l.
Two central anti-parallel P-sheets, which lie roughly perpendicular to each other, and two irregular loops support a large, partially exposed, hydrophobic surface which defines a putative binding site (Fig. 2). The disulfide bridges lie approximately perpendicular to each other, as predicted from homology based on the X-ray crystallographic structure of the first kringle domain of bovine prothrombin [ 161. A cluster of aromatic residues are solvent-accessible and form the presumptive exposed hydrophobic surface.
The dominant structural features of the module are two anti-parallel P-sheets, a small double-stranded sheet comprising the amino-terminal third of the sequence and a triple-stranded sheet comprising the carboxy-terminal residues (Fig. 2). Both P-sheets have a right-handed twist and are stacked to enclose a hydrophobic core comprising three highly conserved aromatic residues and the consensus l-3 and 2-4 disuffide bonds. The disulfide bridge between the first and third cysteines links the two P-sheets, and the disuliide bridge between the second and fourth cysteines links two adjacent strands of the larger g-sheet. The turn between the first and second
Type III modules
Over 300 occurrences of type III modules have been found in over 65 proteins, including extracellular ma-
‘We 1 0
Type
II
0
Type
III
Fig. 1. Schematic
C;1\
III~lII~IIIIII q =, v
EDA.
N
6 ED-B
L
2 Gelatin binding
diagram .of the modular structure of a fibronectin dimer. The two subunits are shown with the am’ino termini (N) to the right and the carboxyl termini K) to the left. Regions implicated in matrix assembly (and discussed in the text) are shown on one of the subunits, and sites of alternative splicing and gelatin binding are shown on the other.
215
216
Macromolecular
assemblages Fig. 2. Folding of (a) type I, (b) type II and (c) type Ill modules. The folding of the type III module is shown more schematically so that interactions of the loops that have been demonstrated in different type Ill modules can be shown.
(a)
trix molecules such as iibronectin and tenascin, cytokine receptors and other cell-surface proteins including the receptor for human growth factor, and intracellular proteins involved in muscle filament formation [17e1. TWO structures of type III modules are available: one was solved by multiwavelength anomalous diffraction phasing of the selenomethionyl third type III module of human tenascin expressed in EscberiZbia coli [18**]; the other was determined by NMR of the tenth type III module of human iibronectin expressed in yeast [19**]. The structure of the tenascin type III domain, which has overall dimensions of 40 X 17 x 28% consists of seven j3-strands arranged into two anti-parallel P-sheets, one of four and one of three strands (Fig. 2) [180*]. The folding topology of the libronectin module [lp*] is identical, as are the crystallographically determined topologies of the two extracellular modules of the human growth hormone receptor [ 20*]. Similar folds have also been shown for the D2 domain of CD4 and the bacterial chaperonin PapD. The relevant sequences in CD4 and PapD are not homologous to type III modules, and thus the structures are considered to be related by convergent rather than divergent evolution (18*-J!?*]. The seven ~-strands are labeled A, B, C, C’, E, F and G and form two P-sheets, A-B-E and Cl-C-F-G (Fig. 2). The fold is similar to that of immunoglobulin domains, except that strand C’ is hydrogen-bonded to strand C in the type III module rather than to strand E in the immunoglobulin domain. Both p-sheets have a right-handed twist, and they stack on top of one another to enclose a hydrophobic core.
C
(b)
In the NMR spectra, the lack of long-range interactions for the residues in the loops between strands C and C’ and between strands F and G suggests that these loops are conformationally labile [19**]. The F-G loop is the location of the Arg-Gly-Asp (RGD) sequence which is essential for adhesive activity of the tenth type III module of iibronectin towards the a& integrin receptor [19*-l and of the third type III module of tenascin towards the a$~ integrin receptor [18*-l. Snake venoms contain a number of small, disulfide-rich proteins, the so-called disintegrins, which interact with integrins to block cell adhesion. The RGD motifs of two disintegrins have been shown by NMR to lie at the apices of conformationally flexible loops [21,22]. Thus, two different protein folds can serve as structural frameworks for a flexible, RGDcontaining cell-adhesive loop.
Heparin
C
HCF
Fibronectin ?
Other loops of type III modules have been demonstrated to participate in binding interactions (Fig. 2). In crystals of the human growth hormone-receptor complex, one type III module interacts with the hormone via its A-B and E-F loops, and the second type III module interacts via its F-G and B-C loops [20-l. A major site of heparin binding to fibronectin has been localized by mutagenesis studies to two argininyl residues at the amino terminus of module III-13 [23].
Connectivities
among
modules
Type III modules occur as repeated arrays and, when visualized by electron microscopy, appear as extended and relatively straight rods [ll]. The calculated contribution of each module to the length of the strand is less than the distance between the amino- and carboxy-terminal ends of the isolated module, and the two ends are on the same side of the module [ 18**]. Thus, successive modules must be tilted relative to one another, and it is likely that alternate modules are rotated by 180”. Such an arrangement might serve to reduce the flexibility of type III arrays. Although the modules are independently folded units [24=], arrays of modules interact to form larger structural and functional domains. The adhesive activity of the RGD-containing tenth type III module of fibronectin is enhanced if the tenth module is in an array that includes III-8 and III-9 1251. A monoclonal antibody specific for fibronectin containing the variably expressed ED-B type III module recognizes an epitope in III-7 which is cryptic in an array of III-7, III-8, and III-9 but unmasked when ED-B is inserted between III-7 and III8 [26*]. Conversely, a monoclonal antibody specific for libronectin that lacks ED-B recognizes III-7 and III-8 in an array lacking the inserted ED-B [ 26.1. Recognition of fibronectin by a monoclonal antibody that blocks assembly (see below) requires that the last type I module of the gelatin-binding region (I-9) be contiguous to the first type III module (III-l) and is sensitive to the reduction of disuliides in I-9 [27]. Finally, binding of the amino-terminal region of iibronectin to cellular assembly sites (see below) requires that the first five type I modules (I-l to I-5) be in an array, such that deletion of any one unit results in the loss of binding activity [28].
Features
of fibronectin
required
Assemblv
of fibronectin
Interchain
disulfide
into extracellular
matrix
Mosher
bridge
The deletion of 20 amino acids from the carboxyl terminus of Iibronectin, including the cysteines involved in interchain disuffide bridges, yields monomeric molecules that do not become incorporated in matrix, i.e. only molecules containing the. bridge region are assembled [31]. Structures of the proteolytically derived heptapeptides, (Val-Gln-Cys-Pro-Ile-Glu-Cys)z, which include the bridge region have been determined by NMR in both water and dimethylsulfoxide [32*-l. The structures provide strong evidence that, although the two subunits are chemically linked head-to-tail in an antiparallel fashion (Fig. l), relative to each other the two chains are effectively disposed in a parallel fashion. Thus, the carboxyterminal bridges act as a terminal which holds the two chains together rather than as a linear junction between mutually extending chains. In addition, the cyclic peptide, which is constrained by the two cystine bridges and the proline residues, is conformationally labile, as shown by substantially different conformations in water and dimethylsulfoxide [ 32**].
Amino-terminal
domain
Most of the binding activity that directs fibronectin to as-‘ sembly sites is contained in the amino-terminal 70 kDa region of the fibronectin subunit, in particular within modules I-l to I-5. Although proteolytic fragments or recombinant proteins containing these modules do not assemble into Iibrils, these proteins bind to assembly sites with the same avidity as intact fibronectin and block the binding and assembly of intact fibronectin [27-291. Removal of any of modules I-l to I-5 results in a protein with an ai%niry for assembly sites 7-lOO-fold less than the nonmutant protein [28]. Mutation of a conserved tyrosine in any one of the modules also causes loss of binding affiity, although the decreases are not as pronounced as in the deletion set. Similar results have been obtained for dimeric fibronectin constructs; molecules containing all five amino-terminal type I modules are incorporated into matrix, whereas molecules lacking module I-5 are not [31].
for assemblv
Fibronectin matrix assembly is a multistep process that requires the presence of viable cells [3,4,29,30]. In the first step, fibronectin binds to the cell surface (Fig. 3). This binding is saturable and reversible. In the following steps, interactions between or among fibronectin molecules lead to the formation of insoluble libronectin multimers. In a presumptive final step, the binding site is regenerated. Three kinds of experiments have been done to probe the features of fibronectin that are required for assembly. First, proteolytic fragments of plasma iibronectin have been tested for their ability to bind to assembly sites and interfere with the binding and assembly of Intact fibronectin. Second, monoclonal antibodies to Iibronectin have been tested for their ability to block binding and assembly. Third, altered iibronectins generated by recombinant techniques have been tested for their ability to assembleinto matrix.
The most likely explanation for the requirement of all five amino-terminal type I modujes for binding activity is that the live modules form a single functional unit, so that deletion of one module or disruption of its structure by mutation of a conserved hydrophobic residue disrupts the structure and function of the entire unit. The ftve modules are also thought to function as a unit in the phenomenon of ‘matrix-driven translocation’ [33=]. This is a phenomenon in which interactions between. distinct regions of an artificial extracellular matrix cause the rapid, unidirectional transport of suspended cells or latex particles into previously unpopulated regions of the matrix.
Presumptive
self-interaction
site
Another functional unit of iibronectln which is probably important in assembly comprises modules I-9 and
217
218
Macromolecular
assemblages
I
I
. I
I
-
~
I Muitimer Membrane
I
I
_ I
I
_
^
I Fig. 3. Model of the proposed cycle of assembly. The protamer, arranged as in Fig. 1, binds in an extended conformation to a cell-surface binding site that includes a hypothetical binding molecule (wedge), CL& integrin (paired vertical bars), and already assembled fibronectin molecules. The change from reversibly bound to irreversibly bound protamer is hypothesized to occur in the intersubunit disulfide bridge region, as represented by the open diamond. Localization and regeneration of assembly sites is controlled by intracellular cytoskeletal elements and associated kinases tarrows).
III-1 [ 271. Two monoclonal antibodies to this region in-
hibit assembly of fibronectin. One antibody recognizes a conformationalIy sensitive epitope which requires intact disulfides io I-9 and contiguity of I-9 with III-l. The second antibody recognizes a linear epitope in LI-1, probably in the E-F loop ([27], also see [19-l >. A gelatin-binding fragment of fibronectin containing this functional unit inhibits the binding of fibronectin to cell layers IO-fold better than does a fragment lacking LLI-1 and the two epitopes. The fragment containing III-1 and the two epitopes does not bind to cell layers, suggesting that inhibition results from binding of the fragment to protameric fibronectin rather than to the assembly sites 1271. A peptide based on a sequence in N-1, however, does bind to cell layers and to intact fibronectin, and might represent a cryptic binding
site that becomes functional during matrix assembly and mediates libronectin-fibronectin interactions [34-l. Two observations indicate that the I-g/III-l sequences might not be crucial for assembly. First, III-1 is one of the less conserved regions of fibronectin [lo]. Second, dimeric fibronectin constructs lacking III-1 or I-9 and III-1 do assemble into matrix [ 31,35**].
Cell
adhesion
site
Yet another feature of fibronectin which is probably important for assembly is the region recognized by the cr5p1 cell-adhesion receptor. This region encompasses III-B, IlI-9 and III-lo, the latter containing the RGD-containing F-G loop. Monoclonal antibodies to this region of
Assembly
libronectin inhibit elaboration of Iibronectin-containing fibrils by cultured fibroblasts [36]. Also, monoclonal antibodies to a5pI block assembly, as described below. Yet, surprisingly, deletion of the RGD sequence for dimeric fibronectin constructs does not interfere with their incorporation into matrix [31].
Carboxy-terminal
modules
When the carboxy-terminal type 1 module regions are fused as a recombinant protein with the amino-terminal modules, the resulting dimeric protein is highly capable of assembling into matrix [35=*]. The carboxy-terminal type I modules, even when joined by the carboxy-terminal disuffide bridges, do not become incorporated into matrix [37]. Mutation or deletion of the carboxy-terminal type I modules in full-length iibronectin does not nevertheless impair grossly the abilities of the altered proteins to be assembled into iibrils [38**]. It is likely, therefore, that the carboxy-terminal type I modules are not involved signiiicantly in assembly.
Features of the cell surface assembly
required
for
The molecules at assembly sites that mediate binding of the amino-terminal modules of fibronectin are not known. Nothing from detergent extracts of iibroblasts competent in assembly binds to an affinity column containing the amino-terminal fragment [39]. Monoclonal antibodies to the as& integrin inhibit binding and assembly of iibronectin by cells [39,40], but also inhibit binding of the amino-terminal ilbronectin fragment, even though there is no evidence that the integrin binds to the fragment [39]. The inhibitory effect of the antibody is evident only after a 10-15 min incubation period. These results suggest that integrins are required for expression of the binding sites for the amino-terminal modules of fibronectin but do not interact with the modules per se. Consistent with this idea, over-expression of a results in increased deposition of Iibronectin matrix r411. Cross-linking studies with transglutaminase and chemical cross-linkers have also been performed [42,43]. Both approaches demonstrate prominent cross-linking of bound amino-terminal fragment to intact iibronectin. Chemical cross-linking studies also identify an - 120 kDa protein which is not aspI [43]. Summing up, then, the only molecule known for sure to interact with the aminoterminal region of fibronectin is fibronectin itself. A major problem in the identification of cell-surface molecules important for fibronectin assembly is that the assembly sites are so labile. For example, transforming growth factor-8 causes a rapid gain of assembly sites [441, whereas agents that elevate intracellular CAMP concentration cause rapid loss of assembly sites [45]. Some cell lines require furthermore the presence of serum for efficient matrix formation [8,46-l. The enhancing activity is enriched ln lipoprotein fractions of serum [46*] and
of fibronectin
into extracellular
matrix
Mosher
can be duplicated by low concentrations of lysophosphatidic acid (WJ Checovich, DR Mosher, unpublished data). Cytochalasin [4] and inhibitors of protein kinase C (CE Somers, DF Mosher, unpublished data) cause prompt ( < 5 min in the case of the protein kinase C inhibitors) loss of cell-surface binding sites for fibronectin or its amino-terminal fragments. The effect of the protein kinase C inhibitors is rapidly reversed upon their removal (CE Somers, DF Mosher, unpublished data). These agents which perturb the binding and assembly of’ libronectin also have profound effects of actin-containing stress fibers and the ability of cells to grip the substratum through focal contacts. Lysophosphatidic acid or serum rapidly stimulates stress fiber and focal adhesion formation in serum-starved iibroblasts [47**]. Conversely, inhibitors of protein kinase C reduce stress fiber and focal adhesion formation [48*-l. The determinants of focal adhesion and stress fiber formation are many, including the GTE-binding protein rho [470*], protein kinase C [48==], tyrosine kinases including focal adhesion-associated kinase [49-511, kinase substrates such as paxillin [49], and, of course, integrins. Integrin-mediated adhesive contacts of motile libroblasts on a substratum demonstrate limited fluctuation in size, density and shape [52*-l. The rapidity with which assembly sites are up- and down-regulated suggests that the same machinery is used in a much more dynamic fashion in matrix assembly. Fibroblasts cultured in serum-containing medium can be considered as the equivalent of specialized fibroblast-like cells, called myofibroblasts, which are present in tissues undergoing contraction [53*]. Tissue contraction is a part of normal wound healing. Cultured libroblasts generate tension, and it is the tension-generating cells that assemble fibronectin matrix [53-l. The association between tension and elaboration of matrix is of potential importance clinically. For instance, stretching of glomerular mesangial cells by glomerular hypertension provokes increased production of extracellular matrix molecules i541.
Model
for assembly
Figure 3 presents a model for assembly that tries to account for the observations described above. The assembly site is shown as being controlled by cytoskeletal and focal adhesion molecules linked to & kitegrins. Fibronectin binds reversibly to the site via its amino-terminal type 1 modules. The fibronectin binds to a yet-to-beidentified molecule, to an altered part of already bound iibronectin, or to an altered integrin. . Something then happens so that fibronectin becomes bound irreversibly as part of a large multimer. The multimer resists dissociation by sodium dodecyl sulfate unless a reducing agent is also present. Such behavior can be considered as evidence for a thiol-disuliide exchange resulting in the formation of disuliides among protamers [29,30]. My laboratory has made a considerable effort to identify the newly formed disulfides and had no success
219
220
Macromolecdar
assemblages
at all. Thus, I presently favor the hypothesis that there is a conformatio@ change of polymerizing molecules, leading to protein-protein interactions that resist dissociatibn. The changed conformation is hypothesized to be stabilized by disulfides. A prime candidate for the conformationally labile region is the carboxy-terminal dlsutide bridge (shown as a change from the open circle to the open diamond in Fig. 3). In a presumptive final step, the binding site is regenerated. This is shown as the cytoskeletal array pulling the integrin and amino-terminal binder in the plane of the membrane, thus focusing the assembly machinery on the untethered end of the newly insolubilized protamer. This recycling is presumably the step that is interrupted by protein kinase C inhibitors or anti-integrin antibodies. The model shown in Fig. 3 applies to addition of a libronectin protamer at the end of a growing fibril. There must be a condensation phase to initiate fiber formation, perhaps an alignment of integrins to bring fibronectin molecules in appostion. There must also be fixation of the non-growing end of the fiber so that the fiber can be tensioned.
Concluding
comments
and future
directions
Our current understanding of the fibronectin assembly process is hardly satisfactory, despite the efforts of a number of laboratories. The only parts of libronectin that may be needed for assembly are the 6ve amino-terminal type I modules and the carboxy-terminal disulfide bridges. How would one then explain the evidence for a critical role of aspI? And how would the cell keep track of the growing end of the fibril? None of the published experiments of matrix assembly in cell culture have been performed without the confounding presence of endogenous fibronectin, either intact or as a heterodimer with recombinant protein. The generation of cell lines that provide just such a null background is important to progress, as is the production of informative recombinant proteins. More studies such as the recent demonstration of a periodic distribution of the ED-A module within a fibril [%I are needed to learn whether or not fibronectin is @deed extended within a fibril and, if so, with what overlap. Finally, the long-standing questions of what b&is the amino-terminal modules to assembly sites and what forces are responsible for holding the multimer together need to be answered.
Acknowledgements
Supportedby grants from the NationalInstitutesof Health and the Wisconsin Affiliate of the American Heart Association.
References
and recommended
reading
Papersof particularinterest, published within the annual period of review, have been highlighted as: of special interest . . .. of outstanding interest 1.
PETERSENTE, SKORSTENGAAIUJ K, VIBE-PEDERSENK: Primary Structure of Fiironectin. In Fibronecfin. Edited by Mosher 0. San Diego: Academic Press; 19891-24.
2.
HYNESRO: Fibronectins
3.
CUR WG: The Role of Intermolecular Disulfide Bonding in Deposition of GP140 in the Extracellular Matrix. / Ccl/ Biol 1984, 99:105-114.
4.
BARRY ELR, MOSHERDF: Factor XIII Cross-Linking of Fibronectin at Cellular Matrix Assembly Sites. J Viol u3etn 1988, 263:10464-10469.
5.
PETERSDP, MOSH~?ROF: Localization of Cell Surface Sites Involved in Fibronectin Fibrillogenesis. / Cell Biol 1987, 104:121-130.
6.
ALUO ‘AE, McKEow?+LONGO PJ: Extracellular Matrix Assembly of Cell-Derived and Plasma-Derived Fibronectins by Substrate-Attached Fibroblasts. J Cell P.!ysiol 1988, 135:459-466.
7.
P~IIS DMP, PORTZ IA, FUUENWIDER J, MOSHEH DF: CoAssembly of Plasma and CeUular Fibronectins into FibrUs in Human Fibroblast Cultures. J Ceil Biol 1990, 111:249-256.
8.
SINGER II: Fibronectin-Cytoskeleton Relationships. In Fibronech Edited by Mosher 0. San Diego: Academic Press; 1989:13~161.
9.
PATCHYL: Modular Exchange Principles in Proteins. CLOT Opin SWuc~ Biol 1991, 1:351-361.
New
York: Springer-Vedag; 1990.
10. .
D~SIMONE DW, NORTON PA, HYNES RO: Identification and Characterization of Alternatively Spliced Fibronectin mRNAs Expressed in Early Xenopus Embryos. Dee Biol 1992, 1491357-369. There are now a number of species for which the sequence of Iibronectin is known, including human, mt, cow, and Xenopus, and the comparisons may give important perspectives on features of fibronectln that are critical for its function. 11.
ODIXMATT E, ENCELJ: Physical Properties of Fibronectin. In Fibronecrh Edited by Mosher OF. San Diego: Academic Press; 1989:2545.
EMCH R, ZENHAUSERN F, JOBIN M, TABORELLIM, DESCOIJIXP: Morphological Difference between Fibronectin Sprayed on Mica and on PMMA UIfrumicmsco~ 1992, 4244:1155-1160. This paper, which presents images of fibronectin sprayed on surfaces and studied by scanning force microscopy in air, complements previ. ous rotary shadowing and scanning transmission electron microscopic sNdies [ll]. 12.
.
13.
BARONM, NORMAN0, WILLIS4 C,~PL)ELLID: Structure of the Fibronectin Type 1 Module. Nalure 1990, 345642-646.
14. .
DOWNINGAK, Druscou PC, HARVEYTS, DUDGEONTH, SMITH BO, BARONM, CAMPBEUID: Solution Structure of the Fibrin Binding Finger Domain of Tissue-Me Plasminogen Activator Determined by ‘H Nuclear Magnetic Resonance. / /MO/ Biol 1992, 225:821-833. The overall fold shows a striking similarity to that of module 1-7 of fibronectin [13]. One significant difference between the two molecules is that hydrophobic residues cover the exposed surface of the major P-sheet region. It is suggested chat one face of this region interacts with anoiher part of the tissue-type plasminogen activator. 15. .
CONSTANI-INE
KL,
MADRID
M,
BANW
I
TTIEXER
M,
PATIHY
I, LUNDM: Refined solution Structure and Ligand-Binding
Properties of PDC-109 Domain B: a Collagen-Binding II Domain.J Mol Biol 1992, 223:281-298.
Type
Assembly
of fibronectin
into extracellular
matrix
221
Mosher
The authors present a structure which is more refined than that reported by the same authors previously and which is more like the structure predicted in [ 16). A hydrophobic binding pocket in the structure is perturbed specifically by leucine analogs, suggesting that this pocket is used to recognize leucine. and/or isoleucine-containing sequences in collagen. HOLLAND SK, HAWX K, BLWE CCF: Deriving the Genedc 16. Structure of the Fibronectin Type II Domain from the Prothrombin Kringle I Crystal Structure. EMEO / 1987, 6:1875-1880.
26.
17. .
27.
CHERNOUSOVMA, FOGER’IY FJ, KOTE~ANSKY VE, MOSHER DF: Role of the I-9 and III-1 Modules of Fibronectin In Formation of an Extracellular Fibronectin Matrix. J Biol Ckm 1991, 266:10851-10858.
28.
Sorrw
BOW P, IXQUITLE RF: Proposed Acquisition of an Animal Protein Domain by Bacteria. Proc Null Acad U 5 A 1992, 8989908994. A systematic screen of a protein database turns up over 300 sequences that meet the criteria for a iibronectin type III module in a wide range of proteins, including bacterial carbohydrate-splitting enzymes. Because type III modules are not found in any fungal or plant proteins, it is suggested that the bacterial modules were acquired by horizontal gene transfer from an animal source. This paper provides strong arguments for this sutprisi.ng conclusion. IEWY DJ, HENDRICKSON WA AUKHIL I, ERICKSON HP: Structure l . of a Fibronectin Type III Domain from Tenascin Phased by MAD Analysis of the Selenomethionyl Protein. Science 1992, 258~987-991. The production of a selenomethionine-substituted protein enables the anomalous diffraction signal from Se to be used to solve the crystallographic phase problem. The structure is refined to 1.8.& resolution. Color graphics, especially of a space-iilling model of a possible alignment type 111modules in a repeated army, make reading this article very worthwhile. Complements the dynamic information in [ I9**]. MAIN
AI,
HARWY
TS,
BARON
M, BOYD
J, C~~IPBELL
ID:
The
Three-DimensionaI Structure of the Tenth Type III Module of Fibronectin: an Insight into RGD-Mediated Interactions. Cell 1992, 71:671-678. A more refined structure than that reported by the same authors previously. An analysis based on 36 final structures, calculated using sim. ulated annealing protocols, is presented. This study complements the static information in [ 18**]. The flexible, dynamic nature of two of the loops when viewed in stereo is particularly impressive. 20. DE Vos AM, ULTSCH M, KO%XXOF~ AA: Human Growth Hor. mone and Extracelhtlar Domain of Its Receptor: Crystal Structure of the Complex. Science 1992, 255:306312. This study gives possible insight into protein-protein interactions that involve more than one type III module. The extracellular domain of the receptor contains two type III modules. In the complex, one hormone molecule interacts with two receptor molecules, and all four modules in the complex contribute residues that participate in hormone binding. 21. ALXER MA, LVARUS RA, DENNIS MS, WAGNER G: Solution Structure of Kistrin, a Potent Platelet Aggregation Inhibitor and GPIIb-IIIa Antagonist. Science 1991, 253445-448. 22. SAUDEKV, ATKINSONRA, PELTONJT: Three-Dimensional Stntcture of Echistatln, the Smallest Active RGD Protein. Bio dwmr3t?y 1991, 30:736+7372. ..
BARKALOWFJB, SZHWARZBALIER JE: Localization of the Major Heparin-Binding Site in Fibronectin. 1 Biol Chem 1991, 266:7812-7818. 24. ~NOVICH SV, Novowxwy VV, BREW SA, INGHAM KC: Re. versible Unfolding of an Isolated Hapatin and DNA Binding Fragment, the First Type III Module from Fiironectin. Biocbim Biophvs Acra 1992, 111957-62. Several fragments containing all or part of the first type III module are isolated, and their folding properties are studied by fluorescence spec troscopy and dlfferentiaI scanning caIorimetty. The results indicate that the module can fold independently of neighboring modules and is corn. posed of a stable core flanked by less compact regions. 25. AOTA S, NAW T, YAMADAKM: Characterization of Regions of FIbronectIn besides the ArglnIne-Glycine-Aspartic Acid Sequence Required for Adhesive Function of the Cell-Binding Domain Using Site-DIIected Mutagenesis. / Biol &em 1991, 266:15938-15943. 23.
B,
IEPflINt
A,
&MANN1
G,
SAGltUTi
M,
ZUtDt
L The Inclusion of the Type III Repeat ED-B In the Flbronectin Molecule Generates Conformational ModiEcations that Unmask. a Cryptic Sequence. / Biol C4em 1992, 267~24689-24692. ED-B is highly conserved (100% identity between rat and human) and should not be antlgenlc. This paper solves the mystety of how it is possible to produce an antibody that recognizes fibronectin with ED-B, and calls attention to the importance of the inter-modutar interactions to structure and function.
J, SXWARZBAUER
J, SEUZGUE J, MOSHER DF:
Five
Type
I Modules of Fibronectin Form a Functional Unit that Binds to Fibroblasts and Staphylococcus aweus J Biol C&em 1991, 266:12840-12843. 29.
18.
19.
CARt+EhiOUA
.
MCKEOWN-LONGO
PJ, MOSHER
DF:
The
Assembly
of
tbe
Fi-
bronectin Matrix in Cultured Human Fibroblast Cells. In Fibronecfin. Edited byMosher DF. San Diego: Academic Press; 1989163-179. Mechanisms for Organization of FiMatrix. Cell D#erenliution Deu 1990, 32:439-450.
30.
FOGERTY
31.
S~HWARZBAUER
bronectin
FJ, MOSHER DF:
JE:
Identihcation
of
the
FibronectIn
Se-
quences Required for Assembly of a FibrIIIar Matrix. / Cell Biol 1991, 113:1463-1473.
32. ..
AN SSA, JIMBNEZ-BARBERO J, PETERSEN TE, LUN& M: The Two Polypeptide Chains in Fibronectin Are Joined in AntiparalIel Fashion: NMR Structural Characterization. Biocbemt3ry 1992, -319927-9933. Structures ot me proteoIytIcaIIy derived carboxy-terminal disulhde
bridge region of libronectin are determined in both water and dimethylsulfoxide. The surprising finding that the two subunits are chemically linked head-to-tail in an antiparallel fashion but are structurally parallel might reconcile the apparent discrepancy between electron microscopic evidence for a V-shaped molecule and chemical evidence for an antiparallel array. 33. .
JAW&IA NS, ROSENFEU)L, KHAN MY, DANWEFSW I, NEV&WN SA: Interaction of Fibronectin with Hepatin ln Model Extracelhtlar Matrices: Role of ArgInine Residues and Sulfate Groups. Biocbemr3~y 1991, 30:15381544. This is the last in a series of papers describing ‘matrix-driven transloca~ tion’. The relationship of this phenomenon to matrix assembly Is not proven, but the structure-function correlations are very similar. MOR~AA, RUOS~AH~E: A Fibronectm Self-Assembly Site Invalved in Fibronectin Matrix Assembly: Reconstruction in a Synthetic Peptide. J Cell Eiol 1992, 118:421429. A successful search for a peptide based on the sequence of III-1 that will bind to Iibronectin. The active sequence includes the B-C loop, strand C, the CC’ loop, and strand C’ pig. 2).
34. .
35.
ICHIHARA-TANAKA
K, MAEDA T, TITANI
K, SEKIGUCHI
K
Mat&t
Assembly of Recombinant FibronectIn Polypeptide Consisting of Amino-Terminal 70 kDa and Carboxyl-Terminal 37kD Regions. FEBS Lett 1992, 299:15>158. This study compares metabolism by L cells of recombinant proteins containing each of the two regions alone with a protein contaktktg the two regions Fused together. The fused form is found in extracellular mauix. A major problem with the use of such an approach in thII paper and in [ 31 I is the formation of heterorkmers of intact endogenous iibronectin subunits and the recombinant protein. Thus, it is dklicult to know whether the recombinant protein becomes incotporated Into matrix by virtue of its own attributes or by virtue of the endogenous subunit to which it is linked. ..
36.
MCDONALDJA, QUADE BJ, BROEKE~MANN TJ, IACHANCE R, FORshw K, HASEGAWAE, AKWAMAS: Fibronectbt’s Cell Adhesive Domain and an Amino-Terminal Mattix Assembly Domain Pat-bcipate In Its Assembly In FIIrobIast PeticeUular Matrix. J Biol c%m 1987, 262:2957-2967.
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Macromolecular 37.
assemblages
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SEKICUCHI K: Recombinant Domain of Human FiL Cells. J Biol Umn 1990,
Son;ll~ J, MOSHER DF: Assembly of Fibronectin Molecules with Mutations or Deletions of the CarboxylsTerminal Type I Modules. BicchentLW-j~ 1993. in press. Non-mutant, mutated and deleted rat fibronectins are produced in monkey COS cells, immunodepleted of monkey libronectin. and studied in CUlNreS of human fibroblasts treated with qcloheximide in order to block the synthesis of human fibronectin. Alterations ofi the carboxy-terminal type I modules have little or no effect on assembly. This SNd, exemplifies the tenuous arguments that can be made follcvwing this type of approach, and the critical need for cells that lack endogenous fibronectin, so enabling the secretion and assembly of recombinant proteins to be studied against a null background.
38. ..
Stress Fibers in Response to Growth 70~389-399. Although lysophosphatidic acid is not mentioned found effects of this lysophospholipid on focal stress fibers form a major part of this paper.
BURIUDC~ K, TURNER CE, ROSIER LH: Tyrosine of Paxillin and pp125Fm Accompanies Extracellular Matrixz a Role in Cytoskeletal Biol 1992. 119:893-903.
50.
GI.IA,Y J-L. SHAUO~AY D: Regulation of Focal AdhesionAssociated Protein Tyrosine Kinase by Both Cellular Adhesion and Oncogenic Transformation. Nnlrrre 1992, 358:690-692. KORI\~)ERG I+ EARP HS. PAKSONS JT. SCHAILLR M, JUUANO RL Cell Adhesion or lntegrin Clustering Increases Phosphoryfation of a Focal Adhesion-Associated Tyrosine Kinase. J
40.
AKNAMA SK, Fibronectin ies: Roles in Cytoskeletal
YAMADA SS, CMN WT. YAMADA KM: Analysis of Receptor Function with Monoclonal AntibodCeU Adhesion, Migration, Matrix Assembly and Organization. J Cd BioI 1989, 109:863-875.
51.
41.
GL%YCOIII F, RUOSI~I E: Elevated Levels of the a$, Fibronectin Suppress the Transformed Phenotype of Chinese Hamster Ovary Cells. Cell 1990, 60849-859.
52. ..
42.
BARRY ELR, MOSHER DF: Factor XIIla-Mediated Cross-Linking of Fiironectin to Fibroblast Cell Layers: Cross-Linking of Cellular and Plasma Fibronectin and of Amino-Terminal Fibroneqin Fragments. J Rio/ Uxm 1989, 264:417+il85.
43.
LIMPER AH, QUADE BJ, RANGWAIA TS, MCDONAD Bind Fibronectin’s Matrix 1991, 266:9697-9702.
UJC~I
in the title, the proadhesions and actin
49.
FOGERN FJ, AKIYA,\IA SK, YAMADA KM, MOSHER DF: Inhibition of Binding of Fibronectin to Matrix Assembly Sites by Antilntegrin (a$,) Antibodies. J Cc/I BioI 1990, 111:699-708.
TM, that
Cc/I 1992,
48. WOODS A, COUCHMAN JR: Protein Kinase C IfWOkment in .. Focal Adhesion Formation. J Cell Sci 1992, 101:277-290. Inhibitors of protein kinase C are shown to inhibit strongly the formation of focal adhesions and stress fibers in cells spreading on a fibroncytin-coated substratum. Interestingly, P,-integrin subunits do not disperse from the focal adhesions in response to the inhibitors.
39.
L&HXVCE RM, BIRDENMEIER JA: CeU Surface Molecules Assembly Domain. J BioI
Factors.
Biol dent
Phosphorylation Cell Adhesion to Assembly. J Cd/
1992, 267~23439-23442.
REGEN CM, I-lOR\Y117. AF: Dynamics of p, Integrin-Mediated Adhesive Contacts in Motile Fibroblasts. J Cc/I Rio/ 1992, 119:1347-1359. Detailed time-lapse and immunofluorescence studies of the distribution of PI in motile cells. This sNdy provides important data on the amount of integrin that is left on the substratum versus the amount that is apparently recycled and on the rates of the two processes.
TO~WEK JJ, HAAKS&IA CJ, EDDY RJ. VALIG~~ MB: Fibroblast Contraction Occurs on Release of Tension in Attached Collagen Lattices: Dependency on an Organized Cytoskeleton and Serum. Am! Ret 1992, 232:359-368. An informative model of libroblasts CUlNrd in collagen lattices. When the lattices are attached to the dish, extensive arrangements of colinear actin stress fibers occur and surface-associated tibronectin librils are fomied. These disappear when the lattices are detached from the dish and allowed to contract. 53. .
44.
AUEN.HOFFMANN BL, CRANKSHAW CL, MOSHER DF: Transforming Growth Factor Beta Increases CeU Surface Binding and Assembly of Exogenous (Plasma) Fibronectin by Normal Human Fibroblasts. MO/ CeII Biol 1988, 8:423-242.
45.
AUEN-HOFFMANN BL MOSHER DF: Matrix Assembly Sites for Exogenous Fibronectin Are Decreased on Human Fibroblasts after Treatment with Agent which Increase Intracellular CAMP. J Biol Cbetn 1987, 262:14361-14365.
54.
RISER BL, CORI-ES P. ZHAO X, BERN~IN J. DLILI~R F, NARINS RG: lntraglomerular Pressure and Mcsangial Stretching Stimulate ExtraceUular Matrix Formation in the Rat. ./ C/in ftz~~est 1992, 90:1932-1943.
CHECOV~CH YQ, SCHULI-L RL, MOSHER DF: Lipoproteins Enhance Fibronectin Binding to Adherent CeUs. A,-/e,-iad 7lmmbas 1992, 12:1122-1130. The authors describe several lipoprotein fractions that increase the number of binding sites for libronectin on several different cell [ypes.
55.
D%t\hlBA bronectin Cultures.
46. .
47. ..
RIDLEY AJ, ti Regulates the
A- The Assembly
Small GTP-Binding of Focal Adhesions
Protein and
rho Actin
BJ, Pt%aS DMP: Arrangement of Cellular Fiin Noncollagenous FibrUs in Human Fibroblast J Cdl Sci 1991, 100:605-612.
DF Mosher, Departments University of Wisconsin, 53706, USA
of Medicine 1300 University
and Biomolecular Avenue, Madison,
Chemistry, Wisconsin