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The laminins
Matrix Biology Vol. 14/1994, pp. 2 7 5 - 2 8 1 © 1994 by GustavFischerVerlag,Stuttgart. Jena. New York
The Laminins RUPERT TIMPL and JUDITH C. BROWN Max-Planck-Institut ffir Biochemie,D-82152 Martinsried, Germany.
Abstract Laminins are extracellular matrix proteins which consist of ct, [5 and y chains with molecular masses of 1 4 0 - 4 0 0 kDa. Chain association occurs through a large triple (~-helical coiled-coil domain towards the C-terminus of each chain. Eight genetically distinct laminin chains ((xl, (~2, ot3, [31, [52, [33, y1, y2) and seven different assembly forms (laminins-1 to -7) are known so far. The most extensively characterized laminin- 1 ((~1131y1) shows calcium-dependent self assembly and heterotypic binding to perlecan, nidogen, fibulin-1 and other matrix components. This binding indicates a crucial role in the supramolecular organization of basement membranes. Laminins also possess binding sites for at least six different integrin receptors and are thus involved in many cell-matrix interactions. Such interactions have been shown to be important during embryonic development and for tissue homeostasis and remodelling. Key words: basement membranes, isoforms, laminin, protein ligands, receptors.
Introduction The first laminin was isolated from the matrix of the Engelbreth-Holm-Swarm (EHS)1 tumor and shown to consist of disulfide-linked chains of about 200 and 400 kDa (Timpl et al., 1979). Subsequent studies demonstrated a cross shape for the protein (Fig. la) and the presence of three genetically distinct but homologous chains (Beck et al., 1990; Engel, 1993). Progress in its structural characterization was accompanied by increasing evidence that laminin is involved in many protein-ligand and cell-matrix interactions (Martin and Timpl, 1987). Through 1993 about 3000 publications on various structural and biological aspects of laminin have appeared. During these investigations it also became clear that the EHS tumor-derived laminin is only one member of a large protein family with several different assembly forms and shapes (Fig. 1). To cope with this complexity, a new nomenclature was adopted for the laminins (Burgeson et al., 1994) which will be used throughout this review. In our short overview, we will outline the basic principles that determine domain organization, chain assembly and 1 Abbreviations used: EHS, Engelbreth-Holm-Swarm; EGF, epidermal growth factor; RGD, Arg-Gly-Asp.
shapes of the various laminin isoforms. This will then be extended to the discussion of structure-function relationships and their relevance in a biological context. Since our citations will be restricted to key references, readers are refered to more comprehensive recent reviews on the structure and biology of laminins (Beck et al., 1990; Engel, 1993; Burgeson, 1993; Paulsson, 1993; Kleinman et al., 1993; Ekblom, 1993).
The laminin protein family Crucial evidence for the existence of isoforms of laminin chains different from those found in EHS tumor laminin came from cDNA sequencing (Hunter et al., 1989; Ehrig et al., 1990; Kallunki et al., 1992) and was preceded and accompanied by biosynthetic and biochemical data indicating different assembly forms and different tissue distributions (Martin and Timpl, 1987; Paulsson 1993; Burgeson, 1993). At present, eight different laminin chains and seven different heterotrimeric assembly forms have been well characterized, as illustrated in Fig. 2. The chains can be classified into three groups (c~, [3, y), based on their sequence identity and domain organization. They include the ctl, ct2 and (*3 chains; the [31, [32 and [33 chains and the
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kalinin or nicein (Burgeson, 1993; Marinkovich ct al., 1993), are significantly smaller than their laminin-1 counterparts (140-200kDa), and evidence extists for a subsequent proteolytic processing of all three chains. Complete sequence analysis of the y2 chain demonstrated significant domain deletions in the N-terminal region when compared to y1 (Kallunki et al., 1992), which explains the lack of substantial short arm structures in the electron micrographs of laminin-5 (Burgeson, 1993). Laminins-6 and -7 are considered to consist of [31 (or [32) and y1 chains together with an approximately 200-kDa (,3 chain (Marinkovich et al., 1992), which results in a Y-shaped structure (Fig. It, Burgeson, 1993). Whether the (*3 chain is identical to that of laminin-5 has not, however, been firmly cstablished. Preliminary evidence exists for several more laminin c, and [~ chains, but their complete sequences and assembly forms are not yet known. Their full characterization will certainly increase the number of laminin isoforms. Even though they have not been found so far, forms with two or even three like chains may also exist, as may splice variants. This could greatly increase the number of laminin assembly forms, indicating a complex evolution and adaptation to different functions.
Domain structure and chain association Fig. 1. Typical examples of rotary shadowed electron micrographs of laminin isoforms. (A) Laminin-1 from EHS tumor; (B) complex of nidogen (arrow heads) with laminin-l; (C) Schwannoma laminin similar to laminin-6 or -7; (D) laminin-2 or -4 from human placenta. Bar: 50 nm. y1 and y2 chains. The domain structures of (*1, [31 and y1 will be discussed below. The assembled laminins are denoted either by their chain compositions or by arabic numbers (Fig. 2). Laminin isoforms can differ by one, two or all three constituent chains, but so far only heterotrimeric assembly forms including one of each of the three classes of chains have been identified. By the adopted new nomenclature (Burgeson et al., 1994), laminin-1 (ctl[31y1) denotes the EHS tumor prototype for which the chains were previously referred to as A (400kDa), B1 (220kDa) and B2 (200kDa) (Beck et al., 1990). Laminins-2, -3 and -4 differ from laminin-I by a novel (*2 or [32 chain or both without causing a significant change in the shape of the molecule (Fig. ld). The (*2 chain was previously named merosin or M component, and only a partial C-terminal sequence has been published (Ehrig et al., 1990). It has a size similar to the (*1 chain, but it is apparently cleaved into 300 and 80kDa components, which remain noncovalently associated. The [32 chain; previously referred to as s-laminin, has a slightly lower molecular mass than the [31 chain but a very similar domain structure (Hunter et al., 1989). The (*3, [33 and y2 chains, which together form laminin-5, previously identified as
A multidomain structure was originally demonstrated for laminin-I by electron microscopy and proteolytic fragmentation (Beck et al., 1990; Engel, 1993). These predictions were fully confirmed and refined by completing the sequence analysis of all three chains (Sasaki et al., 1988). Together the data demonstrated that laminin-1 consists of three similar short arms (domains II1-VI), contributed by the N-terminal regions of the ctl, [31 and y1 chains, and a long arm (domains I, II) where all three chains associate over a considerable length (Fig. 3). Typical structural elements of the short arms are 42 EGFlike repeats of about 60 residues, which contain, however, eight cysteines instead of the six usually found in similar repeats in other proteins. These repeats are terminated or interrupted bv seven globular domains of about 200 residues. The N-terminal globules are homologous and represent a novel protein motif. Several internal globules (ctllVa, (*llVb, yllV, Fig. 3) correspond to large loops inserted into an EGF-like repeat. At the center of the cross, all three chains are linked by three disulfide bridges. The sequences then continue for about 600 residues in the ~*helical domains I and II, which have characteristic heptad repeats of the form (abcdefg), where positions a and d are mainly occupied by hydrophobic residues and e and g by charged residues (Beck et al., 1993). These heptad repeats are not perfect, however, and are interrupted in the [31 chain by a short disulfide-linked loop (* (Fig. 3). The [31 and y1 chains terminate at domain I and are connected by a
Minireview: The Laminins
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Laminin-5 Laminin-6 Laminin-7 Fig. 2. Schematic structures and chain compositions of seven different isoforms of laminin. The chains, referred to as ct, [3 and y by a recently adopted nomenclature (Burgeson et al., 1994), were previously A, B1 and B2, respectively. Laminin-1 corresponds to EHS tumor laminin; laminins-2 to -4 have been previously named merosin, s-laminin and s-merosin, respectively; laminin-5 corresponds to kalinin/ nicein; and laminins-6 and -7 to k-laminin. disulfide bridge. The ~xl chain continues for about 1000residues, which are folded into five homologous 150-180-residue G motifs. Similar globular motifs have recently been identified in other matrix proteins such as perlecan and agrin. As shown by electron microscopy and in part by sequence analysis, laminins-2, -3 and -4 have a very similar domain structure. The ct3, [33 and "/2 chains, however, have a number of deletions in the short arms but apparently maintain a long R-helical domain and, in ct3, a G domain. This pattern has emphasized that the R-helical domain is essential for chain assembly and is thus a hallmark of laminin structure. Studies with laminin-1 fragments have demonstrated that the f$1 and "/1 chains readily form a coiled coil, and in the presence of c~l, a triple-stranded coiled-coil structure (Engel, 1993). This process is independent of disulfide bond formation and yields stable configurations with as little as 100residues from the C-terminal region of domain I. Theoretical predictions from interaction potentials between residues e and g of the heptad repeats of adjacent chains have indicated that not all laminin chain combinations should yield stable configurations (Beck et al., 1993).
In fact, these predictions favor heterotrimeric assembly models of the ct, [3, y type found so far for the isoforms (Fig. 2). These predictions are now in the process of experimental verification in several laboratories and may provide essential clues about the restrictions imposed on laminin chain assembly.
Role in supramolecular structure of basement membranes
Laminins are typical components of basement membranes (Martin and Timpl, 1987; Paulsson, 1993) and because of their multidomain structure provide many interaction sites for other constituents. The mapping of some of these sites to distinct domains of laminin-1 was initially accomplished with a defined set of proteolytic fragments (Fig, 3) and more recently with recombinant fragments. Self assembly of laminin-1 into oligomers and large networks was shown in 1985 and has recently been attributed to the three globular domains VI at the N-terminal ends of the short arms (Yurchenco and Cheng, 1993). Self assembly
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Fig. 3. Domain structure, proteolytic fragments and binding properties of laminin-1. Structural motifs include EGF-like repeats (squares), three different kinds of globular domains (circles) and cthelical domains (open bars). Domains are indicated by roman numerals (Sasaki et al., 1988) and elastase (E) and pepsin (P) fragments by dashed lines (Beck et al., 1990). The C-terminal ends of the disulfide-linked ctl, [31 and y1 chains are at the bottom. Arrows indicate possible ways for the formation of ternary complexes mediated by nidogen. Arrow heads denote the positions of RGD sequences, which differ in the mouse and human ctl chains.
requires calcium, but the localization and sequence of the calcium binding sites are not yet known. The formation of laminin networks is also considered to be a major event in the supramolecular organization of basement membranes in situ, although this has not been shown directly. The fact that laminins are often readily extracted from tissues by neutral buffer containing EDTA (Paulsson et al., 1987; Ehrig et al., 1990; Brown et al., 1994) underscores this possibility. Among the heterotypic interactions of laminin-1, that with nidogen seems to be of the highest affinity (KD -- I riM, Fox et al., 1991). Extraction of the laminin-nidogen complex with EDTA demonstrated lack of calcium dependence (Paulsson et al., 1987), and electron microscopy revealed binding to one of the short arms in a 1:1 stoichiometry (Fig. lb). The nidogen-binding site was recently assigned to the fourth EGF-like repeat of domain llI in the ,/1 chain (Mayer et al., 1993; see Fig. 3). A similar affinity and
specificity of nidogen binding was observed for laminins-2 and -4 which share the y1 chain (Brown et al., 1994). Very little or no binding activity was found for laminin-5 (R. Timpl and R. Burgeson, unpublished) consistent with a limited sequence identity (77%) of the analogous EGF-like repeat in the ,/2 chain (Kallunki et al., 1992). The laminin-binding site of nidogen has been mapped to its C-terminal globular domain G3, while another domain, G2, is responsible for binding to basement membrane collagen IV. This separation of binding sites faciliates the connection of laminin and collagen IV via nidogen (Fox et al., 1991). The fact that laminin-1 does not bind directly to collagen IV (Aumailley et al., 1989) has led to the concept that nidogen is crucial for connecting the networks of laminin and collagen IV in basement membranes. Similar conclusions come from recent studies with laminins-2 and -4 (Brown et al., 1994). Whether this applies to all isoforms of laminin and collagen IV is, however, so far not known. Laminin-1 has a distinct affinity for heparin mediated by fragment E3, which consists of the (.;4 and G5 domains of the (,1 chain (Fig. 3). Apparently the same site is involved in binding to the heparan sulfate chains of the basement membrane proteoglycan perlecan (Battaglia et al., 1992). In addition, laminin binding to the core protein of perlecan can be mediated by nidogen (Fig. 3). This latter interaction seems to be of particular importance for ct2 chain-containing laminins, which have a lower affinity for heparin (Brown et al., 1994). Another laminin ligand is the rod-like basement membrane protein fibulin-1, which binds to the ctl chain of laminin-1 (Fig. 3). It also binds to laminin-4 but not laminin-2, which have ct2 chains rather than ~xl chains but differ in their [3 chains (Brown et al., 1994). Together these data demonstrate overlapping as well as peculiar binding properties of different laminin isoforms. Other potential interactions remain to be firmly established.
Interactions with cellular receptors Soon after its discovery, laminin was identified as a major cell-adhesive protein (Martin and Timpl, 1987). It is now well established that cell binding occurs via a variety of cellular receptors including integrins and several less-wellcharacterized non-integrin receptors (Kramer et al., 1993). At least six different integrins have been identified which bind to different laminins (Table l). They were identified primarily by using inhibiting antibodies or affinity chromatography and more recently by using cells transfected with individual integrin ct subunits (Delwel et al., 1994). The ct6131 integrin seems to be the major receptor for laminin-1 (Sonnenberg et al., 1990) and promotes cell adhesion and spreading by binding to fragment E8 (Aumailley et al., 1987). A similar specificity was found for the c~7[~1 integrin, which shows a more restricted expres-
Minireview: The Laminins
279
Table I. Specificity of integrin binding to different laminin isoforms and localization of laminin-1 binding sites. Integrin
Binding
c~1~1 ct2131 ot361 0t6~31 et7~l Ctv~3
1 1 5, (2, 4) 1, 2, 4, 5 1 1
Laminin a No binding 2, 4 2, 4 1
Laminin-1 binding fragment6 EIX (short arm) EIX (short arm) E8 (al chain, G domain) E8 (c~l chain, G domain) Pl (c~l chain, short arm) c
a Laminin isoforms as indicated in Fig. 2; brackets denote weaker affinities. Including, in part, our own unpublished data. c Cryptic RGD site in mouse sequence. sion (Kramer et al., 1993). Fragment E8 is also involved in the stimulation of neurite outgrowth in cultured neuronal cells (Edgar et al., 1984). The cell-binding activity of E8 is dependent on conformation and requires the presence of a portion of the long arm coiled coil and of ix1 chain G domains (see Fig. 3). This was interpreted to indicate that a binding site in the G domain needed stabilization by the rod to express full actitivy (Deutzmann et al., 1990). Recent studies with recombinant fragments confirmed these observations and demonstrated an additional cryptic binding site for an unkown {31 integrin, which is masked upon assembly into a native E8 structure (Sung et al., 1993). The integrins vary in their ability to bind different laminin isoforms. The (~6f31 integrin seems to be a promiscous receptor, although it probably only plays a minor role in cell binding to laminins-2 and -4 (Brown et al., 1994). The ct3131 integrin shows preference for laminin-5 (Delwel et al., 1994), whereas the collagen receptors ~1~1 and (~2~1 bind to laminin-1 (Kramer et al., 1993) but not to laminins-2 and -4. These receptors can therefore distinguish between different isoforms (Table I). Murine laminin-1 fragment P1 is also a strongly cell-adhesive substrate (Aumailley et al., 1987), due to exposure of a cryptic RGD site which can be recognized, for example, by the vitronectin receptor Ctv[33 (Aumailley et al., 1990). However, this RGD is not conserved in the human laminin ix1 chain, which possesses another RGD site (Fig. 3). The latter seems not to be recognized by RGD-dependent integrins (Aumailley et al., 1990). Whether such cryptic cell-binding sites of laminin become functional during tissue remodelling and invasion remains to be shown. The dystrophin-dystroglycan complex serves as a nonintegrin laminin receptor and connects laminin to the actin filaments of muscle cells (Ervasti and Campbell, 1993). Similar intracellular interactions are also mediated by integrins and cause cell spreading and cytoskeletal rearrangements. In addition, laminin binding may trigger various intracellular signal transduction pathways (Hynes, 1992) which are currently being examined in several laboratories. These data point to a central role of laminins in the control of cellular activities.
Laminin function in a biological context Laminin is known to be a ubiquitous component of mammalian basement membranes and can also occur in some non-basement-membrane localizations. Recent studies with chain-specific antibodies demonstrated both distinct differences and also codistributions in the basement membranes of different organs and their compartments (Paulsson, 1993). However, since some chains are shared by different laminin isoforms (Fig. 2), immunohistology alone will not identify assembly patterns. There is also evidence that subanatomical structures of individual basement membranes differ in laminin composition. Anchoring filaments at the dermal-epidermal junction contain a disulfide-linked complex of laminin-5 and -6 (Burgeson, 1993), while other laminins are present in the underlying lamina densa. In striated muscles, the laminin [32 chain is restricted to neuromuscular synapses and may thus guide the connection with motoneurons (Hunter et al., 1989). Such differences reflect diverse laminin-integrin interactions (Table I) but also possible difference in supramolecular organization patterns. The binding of cells to laminin has dramatic effects on their phenotype, as indicated from many in vitro studies (Martin and Timpl, 1987; Kleinman et al., 1993). Besides adhesion and spreading, the phenomena observed include mitogenic modulation, cell migration, the guidance of nerve axons, maintenance of differentiated cell phenotypes, and the induction of new expression patterns. These activities could also lead to new organizational patterns for cells. Endothelial cells respond by forming tube-like structures similar to those observed during angiogenesis, and some epithelial cells build duct-like structures. This very probably reflects similar responses in vivo, and the elucidation of the underlying mechanisms will be a challenging task. Laminins may also play a crucial role during development. They are expressed in mouse embryos as early as the two-cell stage and subsequently in all newly formed basement membranes (Dziadek and Timpl, 1985). Studies on the expression of different isoforms and their functions by
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gene targeting are still at their beginning. Some clues a r e already available from organ culture studies. During mouse kidney embryogenesis, mesenchymal cells convert into epithelium and form tubular structures. This process is accompanied by the new expression of laminin ctl chains and integrin 6 subunits, which are both involved in cellular recognition (Ekblom, 1993). Furthermore, antibodies that inhibit binding of the (,6131 integrin to laminin-1 fragment E8 interrupt this developmental change, indicating that this interaction is important for maintaining the epithelial phenotype. Recent data have also demonstrated that antibodies to laminin which block nidogen binding cause similar effects (Ekblom et al., 1994). More insight into the biological role of laminins may come from the study of mutant proteins. Drosophila laminin has been shown to possess cd, ]31 and y1 chains similar to the mammalian counterparts (Kusche-Gullberg et al., 1992). Its embryonic expression pattern is spatially and temporarily regulated, which makes mutant analyses meaningful. The unc-6 gene product of C. elegans comprises a laminin-like protein similar to domains IV and V of the y1 chain and seems to control dorsal and ventral cell migrations (Ishii et al., 1992). Further mutants are known for the human y2 chain and are correlated with skin lesions (Pulkkinen et al., 1994). These approaches will therefore become instrumental in furthering our understanding of the biology of laminins.
References
Aumailley, M., Nurcombe, V., Edgar, D., Paulsson, M. and Timpl, R.: The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites..l. Biol. Chem. 262:11532-11538, 1987. Aumailley, M., Wiedemann, H., Mann, K. and Timpl, R.: Binding of nidogen and the laminin-nidogen complex to basement men> brane collagen type IV. Eur. J, Biochem. 184:241-248, 1989. Aumailley, M., Gerl, M., Sonnenberg, A., Deutzmann, R. and Timpl, R.: Identification of the Arg-Gly-Asp sequence in laminin A chain as a latent cell-binding site being exposed in fragment P1. FEBS Lett. 262: 82-86, 1990. Battaglia, C., Mayer, U., Aumailley, M. and Timpl. R.:Basement membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core. Eur. J. Biochem. 208: 359-366, 1992. Beck, K., Hunter, I. and Engel. J.: Structure and function of laminin: anatomy of a multidomain glycoprotein. FASEB .1.4: 148-160, 1990. Beck, K., Dixon, T. W., Engel, J. and Parry, D. A. D.: Ionic interactions in the coiled-coil domain of laminin determine the specificity of chain assembly. J. Mol. Biol. 231: 311-323, 1993. Brown, J. C., Wiedemann, H. and Timpl, R.: Protein binding and cell adhesion properties of two laminin isoforms (AmBleB2e, AmBlsB2e) from human placenta. J. Cell Sci. 107: 329-338, 1994. Burgeson, R.E.: Dermal-epidermal adhesion in skin. In: Molecu-
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bach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 49-66. Burgeson, R. E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel, J., Kleinman, H., Martin, G. R., Meneguzzi, G., Paulsson, M., Sanes, J., Timpl, R., Tryggvason, K. and Yurchenco, P.D.: A new nomenclature for laminins. Matrix Biol. 5, 209-21 I, 1994. Delwel, G.O., de Melker, A.A., Hogervorst, F., Jaspars, L.H., Fles, D. I.. A., Kuikman, I., Lindblom, A., Paulsson, M., Timpl, R. and Sonnenberg, A.: Distinct and overlapping specificities of the c(3AI31 and ct6A~31 integrins: recognition of lammin isoforms. Mol. Biol. Cell 5: 203-215, 1994. Deutzmann, R., Aumailley, M., Wiedemann, H., Pysny, W., Timpl, R. and Edgar, D.: Cell adhesion, spreading and neurite stimulation by laminin fragment E8 depends on maintenance of secondary and tertiary structure in its rod and globular domain. Eur. J. Biochem. 191: 513-522, 1990. Dziadek, M. and Timpl, R.: Expression of nidogen and laminin m basement membranes during mouse embryogenesis and in teratocarcinoma cells. Dev. Biol. 111 : 372-382, 1985. Edgar, D., Timpl, R. and Theonen, H.: The beparin-binding domain of laminin is responsible for the effects on neurite outgrowth and neuronal survival. EMBO J. 3:1463 1468, 1984. Ehrig, K., Leivo, I., Argraves, W. S., Ruoslahti, E. and Engvall, E.: Merosin, a tissue-specific basement membrane protein, is a laminin-like protein. Proc. Natl. Acad. Sci. USA 87: 3264-3268, 1990. Ekblom, P.: Basement membranes in development. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 359-383. Ekblom, P., Ekblom, M., Fecker, L., Klein, G., Zhang, H.-Y., Kadoya, Y., Chu, M.-L., Mayer, U, and Timpl, R.: Role of mesenchymal nidogen in epithelial basement membrane assembly and in organogenesis. Development, in press. Engel, J.: Structure and function of laminin. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 147- 176. Ervasti, J.M. and Campbell, K.P.: A role for the dystrophinglycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 122: 809-823, 1993. Fox, J. W., Mayer, U., Nischt, R., Aumailley, M., Reinhardt, D., Wiedemann, H., Mann, K., Timpl, R., Krieg, T., Engel, J. and Chu, M.-L.: Recombinant nidogen consists of three globular domains and mediates binding of lalninin to collagen type IV. EMBO J. 10:3137-3146, 1991. Hunter, D. D., Shah, V., Merlie, J. p. and Sanes, J. R.: A laminin like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction. Nature338: 229-234, 1989. Hynes, R.O.: lntegrins: versatility, modulation and signalling m cell adhesion. Ce1169: 11-25, 1992. Ishii, N., Wadsworth, W.G., Stern, B.D., Culotti, J.G. and Hedgecock, E. M.: Unc-6, a laminin-related protein, guides cell and pioneer axon migrations in C elegans. Neuron g: 873-881, 1992. Kallunki, P., Sainio, K., Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck, K., Hirvonen, H., Shows, T. B. and Tryggvason, K.: A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment. J. (.ell Biol. 119: 679-693, 1992.
Minireview: The Laminins Kleinman, H. K., Kibbey, M. C., Schnaper, H. W., Hadley, M. A., Dym, M. and Grant, D.S.: Role of basement membrane in differentiation. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D. H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 309-326. Kramer, R. H., Enenstein, J. and Waleh, N. S.: Integrin structure and ligand specificity in cell-matrix interactions. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 239-265. Kusche-Gullberg, M., Garrison, K., MacKrell, A.J., Fessler, L.I. and Fessler, J.H.: Laminin A chain: expression during Drosophila development and genomic sequence. EMBO J. 11 : 4519-4527, 1992. Marinkovich, M. P., Lunstrum, G. P., Keene, D. R. and Burgeson, R. E.: Tile dermal-epidermal junction of human skin contains a noval laminin variant. J. Cell Biol. 119:695 -703, 1992. Marinkovich, M.P., Verrando, P., Keene, D.R., Meneguzzi, G., Lunstrum, G. P., Ortonne, J. P. and Burgeson, R. E.: Basement membrane proteins kalinin and nicein are structurally and immunologically~ identical. Lab. Invest. 69: 295-299, 1993. Martin, G. R. and Timpl, R.: Laminin and other basement membrane components. Ann. Rev. Cell Biol. 3: 57-85, 1987. Mayer, U., Nischt, R., P6schl. E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y. and Timpl, R.: A single EGF-like motif of laminin is responsible for high affinity nidogen binding. EMBO J. 12: 1879-1885, 1993. Paulsson, M.: Laminin and collagen IV variants and heterogeneity in basement membrane composition. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 177-187.
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Paulsson, M., Aumailley, M., Deutzmann, R., Timpl, R., Beck, K. and Engel, J.: Laminin-nidogen complex: extraction with chelating agents and structural characterization. Eur. J. Biochem. 166: 11-19, 1987. Pulkkinen, L., Christiano, A.M., Airenne, T., Haakana, H., Tryggvason, K. and Uitto, J.: Mutations in the ~'2 chain gene (LAM C2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa. Nature Genet. 6: 293-297, 1994. Sasaki, M., Kleinman, H.K., Huber, H., Deutzmann, R. and Yamada, Y.: Laminin, a multidomain protein: the A chain has a unique globular domain and homology with the basement membrane proteoglycan and the laminin B chains. J. Biol. Chem. 263: 16536-16544, 1988. Sonnenberg, A., Linders, C.J.T., Modderman, P.W., Damsky, C.H., Aumailley, M. and Timpl, R.: lntegrin recognition of different cell-binding fragments of laminin (Pl, E3, E8) and evidence that ct6131 but not (x6134functions as a major receptor for fragment E8.J. Cell Biol. 110: 2145-2155, 1990. Sung, U., O'Rear, J.J. and Yurchenco, P.D.: Cell and heparin binding in the distal long arm of laminin: identification of active and cryptic sites with recombinant and hybrid glycoprotein. J. Cell Biol. 123: 1255-1268, 1993. Timpl, R., Rohde, H., Gehron Robey, P., Rennard, S. I., Foidart, J.M. and Martin, G. R.: Laminin - a glycoprotein from basement membranes. J. Biol. Chem. 254: 9933-9937, 1979. Yurchenco, P.D. and Cheng, Y.-S.: Self-assembly and calciumbinding sites in laminin. A three-arm interaction model. J. Biol. Chem. 268: 17286-17299, 1993. Dr. R. Timpl, Max-Planck-Institut f/.ir Biochemie, D-82153 Martinsried, Germany.
The Laminins RUPERT TIMPL and JUDITH C. BROWN Max-Planck-Institut ffir Biochemie,D-82152 Martinsried, Germany.
Abstract Laminins are extracellular matrix proteins which consist of ct, [5 and y chains with molecular masses of 1 4 0 - 4 0 0 kDa. Chain association occurs through a large triple (~-helical coiled-coil domain towards the C-terminus of each chain. Eight genetically distinct laminin chains ((xl, (~2, ot3, [31, [52, [33, y1, y2) and seven different assembly forms (laminins-1 to -7) are known so far. The most extensively characterized laminin- 1 ((~1131y1) shows calcium-dependent self assembly and heterotypic binding to perlecan, nidogen, fibulin-1 and other matrix components. This binding indicates a crucial role in the supramolecular organization of basement membranes. Laminins also possess binding sites for at least six different integrin receptors and are thus involved in many cell-matrix interactions. Such interactions have been shown to be important during embryonic development and for tissue homeostasis and remodelling. Key words: basement membranes, isoforms, laminin, protein ligands, receptors.
Introduction The first laminin was isolated from the matrix of the Engelbreth-Holm-Swarm (EHS)1 tumor and shown to consist of disulfide-linked chains of about 200 and 400 kDa (Timpl et al., 1979). Subsequent studies demonstrated a cross shape for the protein (Fig. la) and the presence of three genetically distinct but homologous chains (Beck et al., 1990; Engel, 1993). Progress in its structural characterization was accompanied by increasing evidence that laminin is involved in many protein-ligand and cell-matrix interactions (Martin and Timpl, 1987). Through 1993 about 3000 publications on various structural and biological aspects of laminin have appeared. During these investigations it also became clear that the EHS tumor-derived laminin is only one member of a large protein family with several different assembly forms and shapes (Fig. 1). To cope with this complexity, a new nomenclature was adopted for the laminins (Burgeson et al., 1994) which will be used throughout this review. In our short overview, we will outline the basic principles that determine domain organization, chain assembly and 1 Abbreviations used: EHS, Engelbreth-Holm-Swarm; EGF, epidermal growth factor; RGD, Arg-Gly-Asp.
shapes of the various laminin isoforms. This will then be extended to the discussion of structure-function relationships and their relevance in a biological context. Since our citations will be restricted to key references, readers are refered to more comprehensive recent reviews on the structure and biology of laminins (Beck et al., 1990; Engel, 1993; Burgeson, 1993; Paulsson, 1993; Kleinman et al., 1993; Ekblom, 1993).
The laminin protein family Crucial evidence for the existence of isoforms of laminin chains different from those found in EHS tumor laminin came from cDNA sequencing (Hunter et al., 1989; Ehrig et al., 1990; Kallunki et al., 1992) and was preceded and accompanied by biosynthetic and biochemical data indicating different assembly forms and different tissue distributions (Martin and Timpl, 1987; Paulsson 1993; Burgeson, 1993). At present, eight different laminin chains and seven different heterotrimeric assembly forms have been well characterized, as illustrated in Fig. 2. The chains can be classified into three groups (c~, [3, y), based on their sequence identity and domain organization. They include the ctl, ct2 and (*3 chains; the [31, [32 and [33 chains and the
276
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a
b
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kalinin or nicein (Burgeson, 1993; Marinkovich ct al., 1993), are significantly smaller than their laminin-1 counterparts (140-200kDa), and evidence extists for a subsequent proteolytic processing of all three chains. Complete sequence analysis of the y2 chain demonstrated significant domain deletions in the N-terminal region when compared to y1 (Kallunki et al., 1992), which explains the lack of substantial short arm structures in the electron micrographs of laminin-5 (Burgeson, 1993). Laminins-6 and -7 are considered to consist of [31 (or [32) and y1 chains together with an approximately 200-kDa (,3 chain (Marinkovich et al., 1992), which results in a Y-shaped structure (Fig. It, Burgeson, 1993). Whether the (*3 chain is identical to that of laminin-5 has not, however, been firmly cstablished. Preliminary evidence exists for several more laminin c, and [~ chains, but their complete sequences and assembly forms are not yet known. Their full characterization will certainly increase the number of laminin isoforms. Even though they have not been found so far, forms with two or even three like chains may also exist, as may splice variants. This could greatly increase the number of laminin assembly forms, indicating a complex evolution and adaptation to different functions.
Domain structure and chain association Fig. 1. Typical examples of rotary shadowed electron micrographs of laminin isoforms. (A) Laminin-1 from EHS tumor; (B) complex of nidogen (arrow heads) with laminin-l; (C) Schwannoma laminin similar to laminin-6 or -7; (D) laminin-2 or -4 from human placenta. Bar: 50 nm. y1 and y2 chains. The domain structures of (*1, [31 and y1 will be discussed below. The assembled laminins are denoted either by their chain compositions or by arabic numbers (Fig. 2). Laminin isoforms can differ by one, two or all three constituent chains, but so far only heterotrimeric assembly forms including one of each of the three classes of chains have been identified. By the adopted new nomenclature (Burgeson et al., 1994), laminin-1 (ctl[31y1) denotes the EHS tumor prototype for which the chains were previously referred to as A (400kDa), B1 (220kDa) and B2 (200kDa) (Beck et al., 1990). Laminins-2, -3 and -4 differ from laminin-I by a novel (*2 or [32 chain or both without causing a significant change in the shape of the molecule (Fig. ld). The (*2 chain was previously named merosin or M component, and only a partial C-terminal sequence has been published (Ehrig et al., 1990). It has a size similar to the (*1 chain, but it is apparently cleaved into 300 and 80kDa components, which remain noncovalently associated. The [32 chain; previously referred to as s-laminin, has a slightly lower molecular mass than the [31 chain but a very similar domain structure (Hunter et al., 1989). The (*3, [33 and y2 chains, which together form laminin-5, previously identified as
A multidomain structure was originally demonstrated for laminin-I by electron microscopy and proteolytic fragmentation (Beck et al., 1990; Engel, 1993). These predictions were fully confirmed and refined by completing the sequence analysis of all three chains (Sasaki et al., 1988). Together the data demonstrated that laminin-1 consists of three similar short arms (domains II1-VI), contributed by the N-terminal regions of the ctl, [31 and y1 chains, and a long arm (domains I, II) where all three chains associate over a considerable length (Fig. 3). Typical structural elements of the short arms are 42 EGFlike repeats of about 60 residues, which contain, however, eight cysteines instead of the six usually found in similar repeats in other proteins. These repeats are terminated or interrupted bv seven globular domains of about 200 residues. The N-terminal globules are homologous and represent a novel protein motif. Several internal globules (ctllVa, (*llVb, yllV, Fig. 3) correspond to large loops inserted into an EGF-like repeat. At the center of the cross, all three chains are linked by three disulfide bridges. The sequences then continue for about 600 residues in the ~*helical domains I and II, which have characteristic heptad repeats of the form (abcdefg), where positions a and d are mainly occupied by hydrophobic residues and e and g by charged residues (Beck et al., 1993). These heptad repeats are not perfect, however, and are interrupted in the [31 chain by a short disulfide-linked loop (* (Fig. 3). The [31 and y1 chains terminate at domain I and are connected by a
Minireview: The Laminins
277
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Laminin-5 Laminin-6 Laminin-7 Fig. 2. Schematic structures and chain compositions of seven different isoforms of laminin. The chains, referred to as ct, [3 and y by a recently adopted nomenclature (Burgeson et al., 1994), were previously A, B1 and B2, respectively. Laminin-1 corresponds to EHS tumor laminin; laminins-2 to -4 have been previously named merosin, s-laminin and s-merosin, respectively; laminin-5 corresponds to kalinin/ nicein; and laminins-6 and -7 to k-laminin. disulfide bridge. The ~xl chain continues for about 1000residues, which are folded into five homologous 150-180-residue G motifs. Similar globular motifs have recently been identified in other matrix proteins such as perlecan and agrin. As shown by electron microscopy and in part by sequence analysis, laminins-2, -3 and -4 have a very similar domain structure. The ct3, [33 and "/2 chains, however, have a number of deletions in the short arms but apparently maintain a long R-helical domain and, in ct3, a G domain. This pattern has emphasized that the R-helical domain is essential for chain assembly and is thus a hallmark of laminin structure. Studies with laminin-1 fragments have demonstrated that the f$1 and "/1 chains readily form a coiled coil, and in the presence of c~l, a triple-stranded coiled-coil structure (Engel, 1993). This process is independent of disulfide bond formation and yields stable configurations with as little as 100residues from the C-terminal region of domain I. Theoretical predictions from interaction potentials between residues e and g of the heptad repeats of adjacent chains have indicated that not all laminin chain combinations should yield stable configurations (Beck et al., 1993).
In fact, these predictions favor heterotrimeric assembly models of the ct, [3, y type found so far for the isoforms (Fig. 2). These predictions are now in the process of experimental verification in several laboratories and may provide essential clues about the restrictions imposed on laminin chain assembly.
Role in supramolecular structure of basement membranes
Laminins are typical components of basement membranes (Martin and Timpl, 1987; Paulsson, 1993) and because of their multidomain structure provide many interaction sites for other constituents. The mapping of some of these sites to distinct domains of laminin-1 was initially accomplished with a defined set of proteolytic fragments (Fig, 3) and more recently with recombinant fragments. Self assembly of laminin-1 into oligomers and large networks was shown in 1985 and has recently been attributed to the three globular domains VI at the N-terminal ends of the short arms (Yurchenco and Cheng, 1993). Self assembly
278
R. Timpl an J. C. Brown
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Fig. 3. Domain structure, proteolytic fragments and binding properties of laminin-1. Structural motifs include EGF-like repeats (squares), three different kinds of globular domains (circles) and cthelical domains (open bars). Domains are indicated by roman numerals (Sasaki et al., 1988) and elastase (E) and pepsin (P) fragments by dashed lines (Beck et al., 1990). The C-terminal ends of the disulfide-linked ctl, [31 and y1 chains are at the bottom. Arrows indicate possible ways for the formation of ternary complexes mediated by nidogen. Arrow heads denote the positions of RGD sequences, which differ in the mouse and human ctl chains.
requires calcium, but the localization and sequence of the calcium binding sites are not yet known. The formation of laminin networks is also considered to be a major event in the supramolecular organization of basement membranes in situ, although this has not been shown directly. The fact that laminins are often readily extracted from tissues by neutral buffer containing EDTA (Paulsson et al., 1987; Ehrig et al., 1990; Brown et al., 1994) underscores this possibility. Among the heterotypic interactions of laminin-1, that with nidogen seems to be of the highest affinity (KD -- I riM, Fox et al., 1991). Extraction of the laminin-nidogen complex with EDTA demonstrated lack of calcium dependence (Paulsson et al., 1987), and electron microscopy revealed binding to one of the short arms in a 1:1 stoichiometry (Fig. lb). The nidogen-binding site was recently assigned to the fourth EGF-like repeat of domain llI in the ,/1 chain (Mayer et al., 1993; see Fig. 3). A similar affinity and
specificity of nidogen binding was observed for laminins-2 and -4 which share the y1 chain (Brown et al., 1994). Very little or no binding activity was found for laminin-5 (R. Timpl and R. Burgeson, unpublished) consistent with a limited sequence identity (77%) of the analogous EGF-like repeat in the ,/2 chain (Kallunki et al., 1992). The laminin-binding site of nidogen has been mapped to its C-terminal globular domain G3, while another domain, G2, is responsible for binding to basement membrane collagen IV. This separation of binding sites faciliates the connection of laminin and collagen IV via nidogen (Fox et al., 1991). The fact that laminin-1 does not bind directly to collagen IV (Aumailley et al., 1989) has led to the concept that nidogen is crucial for connecting the networks of laminin and collagen IV in basement membranes. Similar conclusions come from recent studies with laminins-2 and -4 (Brown et al., 1994). Whether this applies to all isoforms of laminin and collagen IV is, however, so far not known. Laminin-1 has a distinct affinity for heparin mediated by fragment E3, which consists of the (.;4 and G5 domains of the (,1 chain (Fig. 3). Apparently the same site is involved in binding to the heparan sulfate chains of the basement membrane proteoglycan perlecan (Battaglia et al., 1992). In addition, laminin binding to the core protein of perlecan can be mediated by nidogen (Fig. 3). This latter interaction seems to be of particular importance for ct2 chain-containing laminins, which have a lower affinity for heparin (Brown et al., 1994). Another laminin ligand is the rod-like basement membrane protein fibulin-1, which binds to the ctl chain of laminin-1 (Fig. 3). It also binds to laminin-4 but not laminin-2, which have ct2 chains rather than ~xl chains but differ in their [3 chains (Brown et al., 1994). Together these data demonstrate overlapping as well as peculiar binding properties of different laminin isoforms. Other potential interactions remain to be firmly established.
Interactions with cellular receptors Soon after its discovery, laminin was identified as a major cell-adhesive protein (Martin and Timpl, 1987). It is now well established that cell binding occurs via a variety of cellular receptors including integrins and several less-wellcharacterized non-integrin receptors (Kramer et al., 1993). At least six different integrins have been identified which bind to different laminins (Table l). They were identified primarily by using inhibiting antibodies or affinity chromatography and more recently by using cells transfected with individual integrin ct subunits (Delwel et al., 1994). The ct6131 integrin seems to be the major receptor for laminin-1 (Sonnenberg et al., 1990) and promotes cell adhesion and spreading by binding to fragment E8 (Aumailley et al., 1987). A similar specificity was found for the c~7[~1 integrin, which shows a more restricted expres-
Minireview: The Laminins
279
Table I. Specificity of integrin binding to different laminin isoforms and localization of laminin-1 binding sites. Integrin
Binding
c~1~1 ct2131 ot361 0t6~31 et7~l Ctv~3
1 1 5, (2, 4) 1, 2, 4, 5 1 1
Laminin a No binding 2, 4 2, 4 1
Laminin-1 binding fragment6 EIX (short arm) EIX (short arm) E8 (al chain, G domain) E8 (c~l chain, G domain) Pl (c~l chain, short arm) c
a Laminin isoforms as indicated in Fig. 2; brackets denote weaker affinities. Including, in part, our own unpublished data. c Cryptic RGD site in mouse sequence. sion (Kramer et al., 1993). Fragment E8 is also involved in the stimulation of neurite outgrowth in cultured neuronal cells (Edgar et al., 1984). The cell-binding activity of E8 is dependent on conformation and requires the presence of a portion of the long arm coiled coil and of ix1 chain G domains (see Fig. 3). This was interpreted to indicate that a binding site in the G domain needed stabilization by the rod to express full actitivy (Deutzmann et al., 1990). Recent studies with recombinant fragments confirmed these observations and demonstrated an additional cryptic binding site for an unkown {31 integrin, which is masked upon assembly into a native E8 structure (Sung et al., 1993). The integrins vary in their ability to bind different laminin isoforms. The (~6f31 integrin seems to be a promiscous receptor, although it probably only plays a minor role in cell binding to laminins-2 and -4 (Brown et al., 1994). The ct3131 integrin shows preference for laminin-5 (Delwel et al., 1994), whereas the collagen receptors ~1~1 and (~2~1 bind to laminin-1 (Kramer et al., 1993) but not to laminins-2 and -4. These receptors can therefore distinguish between different isoforms (Table I). Murine laminin-1 fragment P1 is also a strongly cell-adhesive substrate (Aumailley et al., 1987), due to exposure of a cryptic RGD site which can be recognized, for example, by the vitronectin receptor Ctv[33 (Aumailley et al., 1990). However, this RGD is not conserved in the human laminin ix1 chain, which possesses another RGD site (Fig. 3). The latter seems not to be recognized by RGD-dependent integrins (Aumailley et al., 1990). Whether such cryptic cell-binding sites of laminin become functional during tissue remodelling and invasion remains to be shown. The dystrophin-dystroglycan complex serves as a nonintegrin laminin receptor and connects laminin to the actin filaments of muscle cells (Ervasti and Campbell, 1993). Similar intracellular interactions are also mediated by integrins and cause cell spreading and cytoskeletal rearrangements. In addition, laminin binding may trigger various intracellular signal transduction pathways (Hynes, 1992) which are currently being examined in several laboratories. These data point to a central role of laminins in the control of cellular activities.
Laminin function in a biological context Laminin is known to be a ubiquitous component of mammalian basement membranes and can also occur in some non-basement-membrane localizations. Recent studies with chain-specific antibodies demonstrated both distinct differences and also codistributions in the basement membranes of different organs and their compartments (Paulsson, 1993). However, since some chains are shared by different laminin isoforms (Fig. 2), immunohistology alone will not identify assembly patterns. There is also evidence that subanatomical structures of individual basement membranes differ in laminin composition. Anchoring filaments at the dermal-epidermal junction contain a disulfide-linked complex of laminin-5 and -6 (Burgeson, 1993), while other laminins are present in the underlying lamina densa. In striated muscles, the laminin [32 chain is restricted to neuromuscular synapses and may thus guide the connection with motoneurons (Hunter et al., 1989). Such differences reflect diverse laminin-integrin interactions (Table I) but also possible difference in supramolecular organization patterns. The binding of cells to laminin has dramatic effects on their phenotype, as indicated from many in vitro studies (Martin and Timpl, 1987; Kleinman et al., 1993). Besides adhesion and spreading, the phenomena observed include mitogenic modulation, cell migration, the guidance of nerve axons, maintenance of differentiated cell phenotypes, and the induction of new expression patterns. These activities could also lead to new organizational patterns for cells. Endothelial cells respond by forming tube-like structures similar to those observed during angiogenesis, and some epithelial cells build duct-like structures. This very probably reflects similar responses in vivo, and the elucidation of the underlying mechanisms will be a challenging task. Laminins may also play a crucial role during development. They are expressed in mouse embryos as early as the two-cell stage and subsequently in all newly formed basement membranes (Dziadek and Timpl, 1985). Studies on the expression of different isoforms and their functions by
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R. Timpl an J. C. Brown
gene targeting are still at their beginning. Some clues a r e already available from organ culture studies. During mouse kidney embryogenesis, mesenchymal cells convert into epithelium and form tubular structures. This process is accompanied by the new expression of laminin ctl chains and integrin 6 subunits, which are both involved in cellular recognition (Ekblom, 1993). Furthermore, antibodies that inhibit binding of the (,6131 integrin to laminin-1 fragment E8 interrupt this developmental change, indicating that this interaction is important for maintaining the epithelial phenotype. Recent data have also demonstrated that antibodies to laminin which block nidogen binding cause similar effects (Ekblom et al., 1994). More insight into the biological role of laminins may come from the study of mutant proteins. Drosophila laminin has been shown to possess cd, ]31 and y1 chains similar to the mammalian counterparts (Kusche-Gullberg et al., 1992). Its embryonic expression pattern is spatially and temporarily regulated, which makes mutant analyses meaningful. The unc-6 gene product of C. elegans comprises a laminin-like protein similar to domains IV and V of the y1 chain and seems to control dorsal and ventral cell migrations (Ishii et al., 1992). Further mutants are known for the human y2 chain and are correlated with skin lesions (Pulkkinen et al., 1994). These approaches will therefore become instrumental in furthering our understanding of the biology of laminins.
References
Aumailley, M., Nurcombe, V., Edgar, D., Paulsson, M. and Timpl, R.: The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites..l. Biol. Chem. 262:11532-11538, 1987. Aumailley, M., Wiedemann, H., Mann, K. and Timpl, R.: Binding of nidogen and the laminin-nidogen complex to basement men> brane collagen type IV. Eur. J, Biochem. 184:241-248, 1989. Aumailley, M., Gerl, M., Sonnenberg, A., Deutzmann, R. and Timpl, R.: Identification of the Arg-Gly-Asp sequence in laminin A chain as a latent cell-binding site being exposed in fragment P1. FEBS Lett. 262: 82-86, 1990. Battaglia, C., Mayer, U., Aumailley, M. and Timpl. R.:Basement membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core. Eur. J. Biochem. 208: 359-366, 1992. Beck, K., Hunter, I. and Engel. J.: Structure and function of laminin: anatomy of a multidomain glycoprotein. FASEB .1.4: 148-160, 1990. Beck, K., Dixon, T. W., Engel, J. and Parry, D. A. D.: Ionic interactions in the coiled-coil domain of laminin determine the specificity of chain assembly. J. Mol. Biol. 231: 311-323, 1993. Brown, J. C., Wiedemann, H. and Timpl, R.: Protein binding and cell adhesion properties of two laminin isoforms (AmBleB2e, AmBlsB2e) from human placenta. J. Cell Sci. 107: 329-338, 1994. Burgeson, R.E.: Dermal-epidermal adhesion in skin. In: Molecu-
lar and Cellular Aspects of Basement Membranes, ed. by Rohr-
bach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 49-66. Burgeson, R. E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel, J., Kleinman, H., Martin, G. R., Meneguzzi, G., Paulsson, M., Sanes, J., Timpl, R., Tryggvason, K. and Yurchenco, P.D.: A new nomenclature for laminins. Matrix Biol. 5, 209-21 I, 1994. Delwel, G.O., de Melker, A.A., Hogervorst, F., Jaspars, L.H., Fles, D. I.. A., Kuikman, I., Lindblom, A., Paulsson, M., Timpl, R. and Sonnenberg, A.: Distinct and overlapping specificities of the c(3AI31 and ct6A~31 integrins: recognition of lammin isoforms. Mol. Biol. Cell 5: 203-215, 1994. Deutzmann, R., Aumailley, M., Wiedemann, H., Pysny, W., Timpl, R. and Edgar, D.: Cell adhesion, spreading and neurite stimulation by laminin fragment E8 depends on maintenance of secondary and tertiary structure in its rod and globular domain. Eur. J. Biochem. 191: 513-522, 1990. Dziadek, M. and Timpl, R.: Expression of nidogen and laminin m basement membranes during mouse embryogenesis and in teratocarcinoma cells. Dev. Biol. 111 : 372-382, 1985. Edgar, D., Timpl, R. and Theonen, H.: The beparin-binding domain of laminin is responsible for the effects on neurite outgrowth and neuronal survival. EMBO J. 3:1463 1468, 1984. Ehrig, K., Leivo, I., Argraves, W. S., Ruoslahti, E. and Engvall, E.: Merosin, a tissue-specific basement membrane protein, is a laminin-like protein. Proc. Natl. Acad. Sci. USA 87: 3264-3268, 1990. Ekblom, P.: Basement membranes in development. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 359-383. Ekblom, P., Ekblom, M., Fecker, L., Klein, G., Zhang, H.-Y., Kadoya, Y., Chu, M.-L., Mayer, U, and Timpl, R.: Role of mesenchymal nidogen in epithelial basement membrane assembly and in organogenesis. Development, in press. Engel, J.: Structure and function of laminin. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 147- 176. Ervasti, J.M. and Campbell, K.P.: A role for the dystrophinglycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 122: 809-823, 1993. Fox, J. W., Mayer, U., Nischt, R., Aumailley, M., Reinhardt, D., Wiedemann, H., Mann, K., Timpl, R., Krieg, T., Engel, J. and Chu, M.-L.: Recombinant nidogen consists of three globular domains and mediates binding of lalninin to collagen type IV. EMBO J. 10:3137-3146, 1991. Hunter, D. D., Shah, V., Merlie, J. p. and Sanes, J. R.: A laminin like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction. Nature338: 229-234, 1989. Hynes, R.O.: lntegrins: versatility, modulation and signalling m cell adhesion. Ce1169: 11-25, 1992. Ishii, N., Wadsworth, W.G., Stern, B.D., Culotti, J.G. and Hedgecock, E. M.: Unc-6, a laminin-related protein, guides cell and pioneer axon migrations in C elegans. Neuron g: 873-881, 1992. Kallunki, P., Sainio, K., Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck, K., Hirvonen, H., Shows, T. B. and Tryggvason, K.: A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment. J. (.ell Biol. 119: 679-693, 1992.
Minireview: The Laminins Kleinman, H. K., Kibbey, M. C., Schnaper, H. W., Hadley, M. A., Dym, M. and Grant, D.S.: Role of basement membrane in differentiation. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D. H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 309-326. Kramer, R. H., Enenstein, J. and Waleh, N. S.: Integrin structure and ligand specificity in cell-matrix interactions. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 239-265. Kusche-Gullberg, M., Garrison, K., MacKrell, A.J., Fessler, L.I. and Fessler, J.H.: Laminin A chain: expression during Drosophila development and genomic sequence. EMBO J. 11 : 4519-4527, 1992. Marinkovich, M. P., Lunstrum, G. P., Keene, D. R. and Burgeson, R. E.: Tile dermal-epidermal junction of human skin contains a noval laminin variant. J. Cell Biol. 119:695 -703, 1992. Marinkovich, M.P., Verrando, P., Keene, D.R., Meneguzzi, G., Lunstrum, G. P., Ortonne, J. P. and Burgeson, R. E.: Basement membrane proteins kalinin and nicein are structurally and immunologically~ identical. Lab. Invest. 69: 295-299, 1993. Martin, G. R. and Timpl, R.: Laminin and other basement membrane components. Ann. Rev. Cell Biol. 3: 57-85, 1987. Mayer, U., Nischt, R., P6schl. E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y. and Timpl, R.: A single EGF-like motif of laminin is responsible for high affinity nidogen binding. EMBO J. 12: 1879-1885, 1993. Paulsson, M.: Laminin and collagen IV variants and heterogeneity in basement membrane composition. In: Molecular and Cellular Aspects of Basement Membranes, ed. by Rohrbach, D.H. and Timpl, R., Academic Press, San Diego, CA, 1993, pp. 177-187.
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