Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA
(e-mail: Roumen.Pankov{at}nih.gov and Kenneth.Yamada{at}nih.gov)
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Introduction |
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FN usually exists as a dimer composed of two nearly identical 250 kDa
subunits linked covalently near their C-termini by a pair of disulfide bonds
(see poster). Each monomer consists of three types of repeating units (termed
FN repeats): type I (purple rectangles), type II (green octagons) and type III
(red ovals). FN contains 12 type I repeats, two type II repeats and 15-17 type
III repeats, which together account for approximately 90% of the FN sequence.
Type I repeats are about 40 amino-acid residues in length and contain two
disulfide bonds; type II repeats comprise a stretch of approximately 60 amino
acids and two intrachain disulfide bonds; and type III repeats are about 90
residues long without any disulfide bonds. All three types of FN repeat are
also found in other molecules, suggesting that FN evolved through exon
shuffling (Patel et al.,
1987
).
Although FN molecules are the product of a single gene, the resulting
protein can exist in multiple forms that arise from alternative splicing of a
single pre-mRNA that can generate as many as 20 variants in human FN (for
reviews, see ffrench-Constant,
1995; Kosmehl et al.,
1996
) (left panel, Plasma and Cellular fibronectin). A major type
of splicing occurs within the central set of type III repeats (left panel, FN
III7 to FN III15). Exon usage or skipping leads to
inclusion or exclusion of either of two type III repeats EDB (also
termed EIIIB or EDII and located between FN repeats III7 and
III8) and EDA (also called EIIIA or EDI and located between FN
repeats III11 and III12). This `yes or no' type of
splicing of FN ED domains is found in many vertebrates, including
Xenopus, chickens, rats and humans.
A third region of alternative splicing is localized to a non-homologous stretch called the V (variable in length) or IIICS (type III connecting segment) region. The structural variations in this region are more complex and species dependent (left panel, lower four gray boxes). In most species studied to date, except chicken, this region can be either partially or completely included or excluded; for example, in human FN, there can be five different V region variants.1 A fourth type of splicing is found in cartilage, where the predominant form of FN [termed (V+C)-] lacks the entire V region along with the FN III15 and FN I10 repeats. Interestingly, this FN isoform exists not only as a homodimer but also in an unusual monomeric configuration (left panel, Single-chain fibronectins). Recently, another single-chain FN (termed FN2) has been described in zebrafish, together with a FN1 form that is very similar to FNs identified in other vertebrates. The truncated FN2 form results from a fifth type of splicing in zebrafish (left panel, Single-chain fibronectins).
FN is an abundant soluble constituent of plasma (300 µg/ml) and other body fluids and also part of the insoluble extracellular matrix. On the basis of its solubility, FN can be subdivided into two forms soluble plasma FN (pFN) and less-soluble cellular (cFN) FN. Plasma FN is synthesized predominantly in the liver by hepatocytes and shows a relatively simple splicing pattern. The alternatively spliced EDA and EDB domains are almost always absent from plasma FN, although both V0 and V+ are present (left panel, Plasma fibronectin). Cellular FN consists of a much larger and more heterogeneous group of FN isoforms that result from cell-type-specific and species-specific splicing patterns (left panel, Cellular fibronectin). Thus, alternative splicing of precursor mRNA from the single FN gene has the capacity to produce a large number of variants, generating FNs with different cell-adhesive, ligand-binding, and solubility properties that provide a mechanism for cells to precisely alter the composition of the ECM in a developmental and tissue-specific manner.
FN can be a ligand for a dozen members of the integrin receptor family (for
a recent review, see Plow et al.,
2000). Integrins are structurally and functionally related
cell-surface heterodimeric receptors that link the ECM with the intracellular
cytoskeleton. A large number of different integrins bind to FN, including the
classic FN receptor
5ß1 (middle panel,
Integrin interaction sites). Extensive analyses have narrowed down the regions
involved in cell adhesion along the lengthy FN molecule to several minimal
integrin-recognition sequences (middle panel, single amino-acid sequences in
red). The best known of these RGD is located in FN repeat
III10. The recognition of this simple tripeptide sequence is
complex and depends on flanking residues, its three-dimensional presentation
and individual features of the integrin-binding pockets. For example, a second
site in FN repeat III9 (the `synergy site' PHSRN, green) promotes
specific
5ß1 integrin binding to FN,
apparently via interactions with the
5 subunit. However,
binding of the FN receptor
5ß1 to FN is not
restricted only to repeats III9 and III10. It can also
interact with an N-terminal fragment containing repeats I1-9 and
II1,2, which also promotes
5ß1-integrin-mediated cell adhesion.
Interestingly, interaction with this N-terminal region can trigger
integrin-mediated intracellular signals that are distinct from those generated
in response to ligation with the RGD sequence.
A second set of FN sequences, which are bound by the
4ß1 integrin, has also received considerable
attention. Two cell-recognition sequences (LDV and REDV) were originally
identified in the alternatively spliced V region. Both of them are recognized
by
4ß1 and
4ß7. Additional sites recognized by the
4ß1 integrin IDAPS and KLDAPT
are also present in repeats III14 and III5,
respectively (the latter also binds to the
4ß7 integrin). Recently, binding of
4ß1 as well as
9ß1 to an EDGIHEL sequence located within
the alternatively spliced EDA segment has been reported, suggesting a possible
adhesive function for the increased EDA-containing FN species observed during
wound healing (Liao et al.,
2002
).
Elucidation of the sites of integrin binding as well as other functionally
important domains within the FN molecule was greatly facilitated by the early
discovery that all FNs are cleaved only in specific regions when subjected to
limited proteolytic digestion (reviewed by
Mosher, 1989;
Hynes, 1990
). Even a protease
capable of cleaving proteins at many sites (such as pronase) will initially
cleave FN, and it will only do this at highly specific, probably non-folded,
unprotected locations. A simplified scheme of the major proteolytic cleavage
sites is shown in the middle panel (see Major proteolytic digestion sites).
The binding activities of FN are often preserved after such proteolysis and
can be identified within particular fragments.
FN has a remarkably wide variety of functional activities besides binding
to cell surfaces through integrins. It binds to a number of biologically
important molecules that include heparin, collagen/gelatin, and fibrin. These
interactions are mediated by several distinct structural and functional
domains, which have been defined by proteolytic fragmentation or recombinant
DNA analyses (see Mosher,
1989; Hynes, 1990
;
Yamada and Clark, 1996
; and
the website
http://www.gwumc.edu/biochem/ingham/fnpage.htm).
FN contains two major heparin-binding domains that interact with heparan
sulfate proteoglycans (right panel, Ligand interaction sites). The strongest
heparin-binding site is located in the C-terminal part (Heparin II) and a
weaker binding domain is situated at the N-terminal end of the protein
(Heparin I). The high-affinity heparin II domain can also bind to a widely
distributed glycosaminoglycan, chondroitin sulfate, whereas the weaker
heparin-binding domain contains a Staphylococus-aureus-binding site
that mediates FN interactions with bacteria. Recently, a novel
glycosaminoglycan-binding site has been identified within the V region of FN
(Mostafavi-Pour et al., 2001)
(marked as `Heparin' at the V domain). In at least some cell types, the
heparin-binding domains of FN potentiate cell adhesion.
The collagen-binding domain includes repeats I6-9 and
II1,2, and it binds far more effectively to denatured collagen
(gelatin) than to native collagen. Thus, FN interactions with collagens in
general may be due to its binding to unfolded regions of the collagen triple
helix. It has been suggested that the physiological function of the
collagen/gelatin-binding domain is related more to binding and clearance of
denatured collagenous materials from blood and tissue than to mediating cell
adhesion to collagen. Interestingly, however, a recent analysis of the
physiological state of collagen indicates that the triple helix is likely to
unfold locally at body temperature (Leikina
et al., 2002), which suggests that this FN domain could be
involved in interactions with native collagen in vivo.
FN also contains two major fibrin-binding sites (Fibrin I and Fibrin II). The major site is in the N-terminal domain and is formed by type I repeats 4 and 5. The interaction of FN with fibrin is thought to be important for cell adhesion or cell migration into fibrin clots. In both cases, cross-linking between FN and fibrin mediated by factor XIII transglutaminase is proposed to mediate the effect (the cross-linking site on the FN molecule is marked by factor XIIIa and an arrow). The interaction of FN with fibrin may also be involved in macrophage clearance of fibrin from circulation after trauma or in inflammation.
FNs are glycoproteins that contain 4-9% carbohydrate, depending on the cell source. Glycosylation sites that are either N-linked (red stars) or O-linked (green star) reside predominantly within type III repeats and the collagen-binding domain. The physiological role of the carbohydrates is not certain, although they appear to stabilize FN against hydrolysis and modulate its affinity to some substrates.
Although the plasma FN that circulates in blood is in a closed, reportedly
non-active form, most of the FN activities in the body have been ascribed to
the insoluble form of FN that exists as part of the extracellular matrix (see
the immunofluorescence image obtained with anti-FN antibodies at the bottom of
the middle panel labeled Fibronectin-based matrix). The creation and
deposition of insoluble FN fibrils into the ECM is a tightly regulated,
cell-mediated process termed FN fibrillogenesis or FN matrix assembly (for a
review, see Geiger et al.,
2001). A critical step in this process is self-association of FN
into aggregates and fibrils, which is directed by multiple binding sites that
have been identified along the molecule (right panel, Sites involved in
fibronectin fibrillogenesis). Some of these self-interaction sites are exposed
and available for binding (marked in yellow), while others are cryptic (marked
in light brown) and become accessible only after conformational changes, for
example, by cell-driven mechanical stretching of the FN molecule.
FN is one of the largest multi-domain proteins for which domain organization, molecular interactions, and key functions have been established in great detail. Exploration of the cell-type-specific splicing variants, glycosylation patterns and their relationship to health and disease will be further challenges in the study of this fascinating molecule.
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Footnotes |
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References |
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Carsons, S. E. (1989). Fibronectin in Health and Disease. Florida: CRC Press, Inc.
ffrench-Constant, C. (1995). Alternative splicing of fibronectin many different proteins but few different functions. Exp. Cell Res. 221,261 -271.[CrossRef][Medline]
Geiger, B., Bershadsky, A., Pankov, R. and Yamada, K. M. (2001). Transmembrane extracellular matrix-cytoskeleton crosstalk. Nat. Rev. Mol. Cell. Biol. 2, 793-805.[CrossRef][Medline]
George, E. L., Georges-Labouesse, E. N., Patel-King, R. S.,
Rayburn, H. and Hynes, R. O. (1993). Defects in mesoderm,
neural tube and vascular development in mouse embryos lacking fibronectin.
Development 119,1079
-1091.
Hynes, R. O. (1990). Fibronectins. New York: Springer-Verlag.
Kosmehl, H., Berndt, A. and Katenkamp, D. (1996). Molecular variants of fibronectin and laminin: structure, physiological occurrence and histopathological aspects. Virchows Arch. 429,311 -322.[Medline]
Leikina, E., Mertts, M. V., Kuznetsova, N. and Leikin, S.
(2002). Type I collagen is thermally unstable at body
temperature. Proc. Natl. Acad. Sci. USA
99,1314
-1318.
Liao, Y. F., Gotwals, P. J., Koteliansky, V. E., Sheppard, D.
and Van De Water, L. (2002). The EIIIA segment of fibronectin
is a ligand for integrins 9ß1 and
4ß1 providing a novel mechanism for
regulating cell adhesion by alternative splicing. J. Biol.
Chem. 277,14467
-14474.
Liu, X. and Collodi, P. (2002). Novel form of
fibronectin from zebrafish mediates infectious hematopoietic necrosis virus
infection. J. Virol. 76,492
-498.
Mosher, D. F. (1989). Fibronectin. San Diego: Academic Press, Inc.
Mostafavi-Pour, Z., Askari, J. A., Whittard, J. D. and Humphries, M. J. (2001). Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants. Matrix Biol. 20, 63-73.[CrossRef][Medline]
Patel, R. S., Odermatt, E., Schwarzbauer, J. E. and Hynes, R. O. (1987). Organization of the fibronectin gene provides evidence for exon shuffling during evolution. EMBO J. 6,2565 -2572.[Abstract]
Plow, E. F., Haas, T. A., Zhang, L., Loftus, J. and Smith, J.
W. (2000). Ligand binding to integrins. J. Biol.
Chem. 275,21785
-21788.
Yamada, K. M. and Clark, R. A. F. (1996). Provisional matrix. In The Molecular and Cellular Biology of Wound Repair (ed. R. A. F. Clark), pp. 51-93. New York: Plenum Press.
Zhao, Q., Liu, X. and Collodi, P. (2001). Identification and characterization of a novel fibronectin in zebrafish. Exp. Cell Res. 268,211 -219.[CrossRef][Medline]
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