Laminin A


EVOLUTIONARY HOMOLOGS (part 1/3)

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for various Laminin domains.

Drosophila Laminin related proteins

The Drosophila Laminin B2 gene differs substantially in size and exon pattern from those of the human B1 and B2 genes. However, as in the case of the human B1 gene, the overall exon pattern of the Drosophila B2 gene does not correlate well with the highly conserved structural domains and internal repeats of the B2 polypeptide chain. Unlike the human and mouse B1 and B2 genes, the 2.1-kb 5'-flanking region of the Drosophila B2 gene contains a TATA box and two CAAT boxes. Other potential transcriptional regulatory sequences include two reverse complementary cAMP response element sequences, two sequences that are homologous to the retinoic acid response element motifs of the mouse B1 gene, and sequences homologous to the binding sites for transcription factors dFRA, dJRA, Zeste, and possibly GAGA. When transfected into Drosophila SL-2 cells, pCAT plasmid containing 2,090 bp of 5'-flanking region shows a 3.0- to 3.5-fold increase in chloramphenicol acetyltransferase activity after induction with retinoic acid and/or 8-bromo-cAMP. These results suggest that this 5'-flanking promoter region may contain DNA sequences that can regulate the expression of the Laminin B2 gene (Chi, 1991).

It has been shown that the Drosophila proteins encoded by the tumor suppressor fat gene, the neurogenic slit gene and crumbs gene all contain domains homologous with modules identified previously in Laminin A. These proteins share a number of features: they have large extracellular regions containing Laminin A modules linked to epidermal growth factor-like domains, and all are involved in cell-cell interactions that are crucial for correct morphogenesis of ectodermal tissues (development of midline neuroepithelia, organization of epithelial tissues etc.). It is suggested that the Laminin A-type modules of these proteins play important roles in the interactions that control ectodermal differentiation (Patthy, 1992).

The Drosophila homolog of vertebrate classic cadherins is a transmembrane protein with similarity to vertebrate classic cadherins. DE-cadherin is distinguishable from its vertebrate counterparts by a large insertion with local sequence similarity to Fat, Laminin A chain, Slit, and Neurexin I at the proximal region of the extracellular domain (Oda, 1994).

Laminin subunit role in C. elegans development

Laminins are heterotrimeric (alpha/ß/gamma) glycoproteins that form a major polymer within basement membranes. Different alpha, ß and gamma subunits can assemble into various laminin isoforms that have different, but often overlapping, distributions and functions. The contributions of the laminin alpha subunits to the development of C. elegans has been studied. There are two alpha, one ß and one gamma laminin subunit, suggesting two laminin isoforms that differ by their alpha subunit assemble in C. elegans. Near the end of gastrulation and before other basement membrane components are detected, the alpha subunits are secreted between primary tissue layers and become distributed in different patterns to the surfaces of cells. Mutations in either alpha subunit gene cause missing or disrupted extracellular matrix where the protein normally localizes. Cell-cell adhesions are abnormal: in some cases essential cell-cell adhesions are lacking, while in other cases, cells inappropriately adhere to and invade neighboring tissues. Using electron microscopy, adhesion complexes are observed at improper cell surfaces and disoriented cytoskeletal filaments. Cells throughout the animal show defective differentiation, proliferation or migration, suggesting a general disruption of cell-cell signaling. The results suggest a receptor-mediated process localizes each secreted laminin to exposed cell surfaces and that laminin is crucial for organizing the extracellular matrix, surface receptors and intracellular proteins at those surfaces. It is proposed this supramolecular architecture regulates adhesions and signaling between adjacent tissues (Huang, 2003).

This study reveals early events that lead to the assembly of basement membranes in vivo. Both laminin alpha subunit genes are apparently expressed under the control of signals that initiate and regulate gastrulation. Gene expression is first detected in the nuclei of cells that are ingressing through a furrow along the ventral midline and, as the tissue layers begin to be organized, cytoplasmic RNA is detected. At this time, the gene encoding laminin alphaA, lam-3, is expressed in pharyngeal and epidermal cells, and weakly in intestinal cells, whereas the gene encoding laminin alphaB, epi-1, is expressed in intestinal, pharyngeal and myoblast cells. Both laminin alpha subunit proteins are then deposited between the tissue layers. Near the end of embryogenesis, laminin alpha subunit gene expression changes, the laminin alphaA gene being expressed most notably in the pharynx and the laminin alphaB gene in the muscle cells (Huang, 2003).

The distribution of the different laminin subunits is probably a cell-surface receptor-mediated process. Although both laminin proteins are secreted between tissue layers during gastrulation, they do not indiscriminately assemble. Rather, each subunit is distributed in a different pattern to cell surfaces and, furthermore, they are not necessarily associated with the cells that express the subunit. The staining pattern of laminin alphaA along the nerve tracts is revealing because the basement membrane associated with the nerve tracts is not morphologically distinguished from other regions of the epidermal basement membrane. It is hypothesized that laminin alphaA is concentrated at neuronal cell surfaces by specific cell-surface receptor(s) and that the laminin alphaA containing trimer mixes with the alphaB trimer in the basement membrane at these locations. The association of laminin alphaA even when the axons are mispositioned supports this conclusion. Also revealing is the finding that even when the two laminins might appear to be able to intermingle, such as where the pharynx and body wall basement membranes are juxtaposed, they in fact remain separated, indicating that each is anchored to a particular architecture (Huang, 2003).

The laminin alpha subunits associate with cell surfaces before the reported expression of other basement membrane components and they are required to assemble stable basement membranes. Evidence from other systems also suggests that early laminin expression is essential for further basement membrane assembly. For example, antisense experiments have shown that the disruption of laminin alpha subunit expression in cultured cells blocks laminin secretion and prevents the subepithelial accumulation of entactin/nidogen and type IV collagen, and the formation of a basement membrane. In addition, the laminin gamma1 knockout arrests at peri-implantation and neither the embryos nor derived embryoid bodies form basement membrane. In both these cases other basement membrane proteins are detected but only as disorganized extracellular deposits (Huang, 2003 and references therein).

Like the laminin alpha subunits, other extracellular matrix proteins in C. elegans also localize to different basement membranes and are not necessarily associated with the cells that express the protein. This suggests that cell surface-associated molecules are required for the assembly of the extracellular matrix proteins into basement membranes. Collagen IV localizes to all basement membranes except those between the pseudocoelomic cavity and the body wall muscles or the epidermis. Nidogen (entactin), which can bind both collagen IV and laminin with high affinity, is associated with muscle cells as the embryo begins to elongate and subsequently is detected at the pharynx, intestine and gonad primordia. In larvae and adults, nidogen is detected in most basement membranes, but is most strongly detected around the nerve ring and developing gonad. It becomes concentrated at the edges of muscle quadrants and on the sublateral nerves, which run longitudinally along the center margin of the muscle quadrants. This staining pattern is different from either laminin alpha subunit, although there are striking similarities to the laminin alphaA pattern with regard to nervous system staining. Nidogen is found at all locations where collagen IV localizes. The C. elegans homolog of mammalian perlecan, a heparan sulfate proteoglycan that binds nidogen/entactin and laminin, plays an essential role in muscle development and has been shown by antibody staining to localize to basement membranes at the bodywall and anal muscles, and at the pharynx and gonad. Perlecan may be produced by epidermal cells and recruited to the body wall muscles cells (Huang, 2003 and references therein).

The results suggest that although laminin may associate with many different surfaces of cells, it is normally excluded from doing so. In lam-3 mutants, an inappropriate matrix can accumulate between the pharyngeal cells, presumably because defective adhesions between the pharyngeal cells allow laminin inappropriate access to lateral surfaces. Where body wall basement membrane is defective in epi-1 mutants, growth cones, which normally migrate between the body wall basement membrane and the basal surface of the epidermis, may become inadvertently exposed to secreted non-polymerized laminin. As a result, laminin is inappropriately assembled at exposed surfaces all around the axons (Huang, 2003).

Laminin expression

Described here is the isolation and characterization of a cDNA clone encoding a region of the carboxy terminal globular domain (G domain) of the alpha-1 chain of laminin from the sea urchin Strongylocentrotus purpuratus. Sequence analysis indicates that the 1.3 kb cDNA (spLAM-alpha) encodes the complete G2 and G3 subdomains of sea urchin alpha-laminin. The 11 kb spLAM-alpha mRNA is present in the egg and declines slightly in abundance during development to the pluteus larva. The spLAM-alpha gene is also expressed in a variety of adult tissues. Whole mount in situ hybridization of gastrula stage embryos indicates that ectodermal and endodermal epithelia and mesenchyme cells contain the spLAM-alpha mRNA. Immunoprecipitation experiments using an antibody made to a recombinant fusion protein indicates spLAM-alpha protein is synthesized continuously from fertilization as a 420 kDa protein that accumulates from low levels in the egg to elevated levels in the pluteus larva. Light and electron microscopy identify spLAM-alpha as a component of the basal lamina. Blastocoelic microinjection of an antibody to recombinant spLAM-alpha perturbs gastrulation and skeleton formation by primary mesenchyme cells. This suggests an important role for laminin in endodermal and mesodermal morphogenesis (Benson, 1999).

A mammalian recombinant strategy was established to dissect rules of basement membrane laminin assembly and secretion. The alpha-, beta-, and gamma-chain subunits of laminin-1 are expressed in all combinations, transiently and/or stably, in a near-null background. In the absence of its normal partners, the alpha chain is secreted as intact protein and protein that has been cleaved in the coiled-coil domain. In contrast, the beta and gamma chains, whether expressed separately or together, remain intracellular, with formation of betabeta or betagamma, but not gammagamma, disulfide-linked dimers. Secretion of the beta and gamma chains requires simultaneous expression of all three chains and their assembly into alphabetagamma heterotrimers. Epitope-tagged recombinant alpha subunit and recombinant laminin were affinity-purified from the conditioned medium of alphagamma and alphabetagamma clones. Rotary-shadow electron microscopy reveals that the free alpha subunit is a linear structure containing N-terminal and included globules with a foreshortened long arm, while the trimeric species has the typical four-arm morphology of native laminin. It is concluded that the alpha chain can be delivered to the extracellular environment as a single subunit, whereas the beta and gamma chains cannot, and that the alpha chain drives the secretion of the trimeric molecule. Such an alpha-chain-dependent mechanism could allow for the regulation of laminin export into a nascent basement membrane, and might serve an important role in controlling basement membrane formation (Yurchenco, 1997).

Mouse and bovine endothelial cells express a novel 400-kDa laminin alpha chain complexed to beta1 and gamma1 laminin chains. This laminin isoform has been purified from the conditioned medium of a mouse peripheral lymph node endothelial cell line, SVEC. The sequence is identical to that of the laminin alpha5 gene. The laminin alpha5 chain is expressed mainly by epithelial, endothelial, and myogenic cells: In both embryonic and mature tissues the laminin alpha5 chain is strongly expressed by epithelial cells, the bronchi of the lungs and the developing kidney tubules being the sites of strongest expression. However, laminin alpha5 is not associated with early stages of epithelial cell development, but rather with epithelial cell maturation. Widespread expression of laminin alpha5 in endothelial cells is apparent only in tissues of mature mice, its appearance correlating approximately with sexual maturity. During embryogenesis and in newborn tissues, laminin alpha5 occurs in the basement membranes of larger blood vessels only, excluding a role in angiogenic processes. Smooth muscle and skeletal muscle cells are the only other cell types that show considerable laminin alpha5 expression, with skeletal muscle exhibiting a developmentally regulated pattern of expression: the laminin alpha5 chain occurs in skeletal muscle fiber basement membranes early in embryogenesis (E13-E15) but decreases with development, remaining strongly expressed only at the neuromuscular junction. The data show that laminin alpha5 expression is associated with epithelial and endothelial cell maturation, implicating a role for this laminin chain in the maintenance of differentiated epithelial and endothelial cell phenotype (Sorokin, 1997).

The mouse laminin alpha4 chain contains a 24-residue signal peptide preceding the mature alpha4 chain of 1,792 residues. Expression is weak at day 7, but it later increases and peaks at day 15. In adult tissues the strongest expression is observed in the lung and in cardiac and skeletal muscles. Weak expression is also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. Expression of the laminin alpha4 chain is mainly localized to mesenchymal cells. Strong expression is seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression is associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. The laminin alpha4 chain localizes primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium (Iivanainen, 1997).

Laminins are expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and adult mouse knee joint. Messenger RNAs for the alpha 1, alpha 2, beta 1, beta 2, and gamma 1 chains are expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and the articular cartilage cap and epiphyseal growth plate of adult mouse knee joint. Through the use of chain-specific antibodies, staining for laminins is observed in the cytoplasm of chondrocytes from chick embryo sternum, mouse limb bud, and adult mouse knee joint. Cultured chick embryonic sternal chondrocytes express laminin mRNAs in the proliferating stage (2-3 days of culture) but the level increases in the aggregated cells during the maturation stage (5-7 days of culture). Thus, laminins are expressed in significant amounts by chondrocytes and may have an important role in cartilage development (Lee, 1997).

Laminins, heterotrimers of alpha, beta, and gamma chains, are prominent constituents of basal laminae (BL) throughout the body. Previous studies have shown that laminins affect both myogenesis and synaptogenesis in skeletal muscle. The distribution of the 10 known laminin chains has been studied in muscle and peripheral nerve, and the ability of several heterotrimers to affect the outgrowth of motor axons has been assayed. Cultured muscle cells express four different alpha chains (alpha1, alpha2, alpha4, and alpha5), and developing muscles incorporate all four into BL. The portion of the muscle's BL that occupies the synaptic cleft contains at least three alpha chains and two beta chains, but each is regulated differently. Initially, the alpha2, alpha4, alpha5, and beta1 chains are present both extrasynaptically and synaptically, whereas beta2 is restricted to synaptic BL from its first appearance. As development proceeds, alpha2 remains broadly distributed, whereas alpha4 and alpha5 are lost from extrasynaptic BL and beta1 is lost from synaptic BL. In adults, alpha4 is restricted to primary synaptic clefts whereas alpha5 is present in both primary and secondary clefts. Thus, adult extrasynaptic BL are rich in laminin 2 (alpha2beta1gamma1), and synaptic BL contain laminins 4 (alpha2beta2gamma1), 9 (alpha4beta2gamma1), and 11 (alpha5beta2gamma1). Likewise, in cultured muscle cells, alpha2 and beta1 are broadly distributed but alpha5 and beta2 are concentrated at acetylcholine receptor-rich "hot spots," even in the absence of nerves. The endoneurial and perineurial BL of peripheral nerve also contain distinct laminin chains: alpha2, beta1, gamma1, and alpha4, alpha5, beta2, gamma1, respectively. Mutation of the laminin alpha2 or beta2 genes in mice not only leads to loss of the respective chains in both nerve and muscle, but also to coordinate loss and compensatory upregulation of other chains. Notably, loss of beta2 from synaptic BL in beta2(-/-) "knockout" mice is accompanied by loss of alpha5, and decreased levels of alpha2 in dystrophic alpha2(dy/dy) mice are accompanied by compensatory retention of alpha4. Motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (alpha1beta1gamma1) and laminin 2, fail to cross from laminin 4 to laminin 1, and stop on contact with laminin 11. The ability of laminin 11 to serve as a stop signal for growing axons explains, in part, axonal behaviors observed at developing and regenerating synapses in vivo (Patton, 1997).

Basement membranes (BM) are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Analysis of a fibroblast growth factor (FGF) receptor mutation has raised the possibility that protein kinase B (Akt/PKB) activated by FGF is connected to the expression of certain laminin and type IV collagen isotypes. This hypothesis was tested; constitutively active Akt/PKB, an important downstream element of phosphoinositide 3'-kinase signaling, was shown to induce the synthesis of laminin-1 and collagen IV isotypes and cause their translocation to the BM. By using promoter-reporter constructs, constitutively active phosphoinositide 3'-kinase-p110 or Akt/PKB was shown to activate, whereas dominant negative Akt/PKB was shown to inhibit, transcription of laminin beta1 and collagen IV alpha1 in differentiating C2 myoblast- and insulin-induced Chinese hamster ovary-T cell cultures. These results suggest that Akt/PKB activated by receptor tyrosine kinases is involved in the positive regulation of BM formation. Thus, Akt/PKB activates laminin and collagen IV at the level of transcription. Akt/PKB controls numerous transcription factors. Whether the forkhead family, the NFkappa B system, or other mechanisms connect Akt/PKB activation with the transcription of laminin and collagen IV chains remains to be determined. It is tempting, nevertheless, to speculate that this regulation represents the positive side of BM remodeling, whereas metalloproteinases represent its negative side. Such positive regulation of BM formation could result in the local amplification of cell signaling mediated by the various signaling molecules associated with the BM (Li, 2001).

Laminin expression and the subsequent deposition of a basement membrane by primitive endoderm cells is necessary for early mammalian development. The transcription factors COUP-TF I and II are up-regulated in primitive endoderm cells faster than LAMB1 and LAMC1, and either COUP-TF is sufficient to induce expression of these laminin genes (Murray, 2001).

Covalent modification of Laminin

beta 1,4-Galactosyltransferase is unusual among the glycosyltransferases in that a subpopulation exists on the cell surface in addition to its traditional biosynthetic location within the Golgi complex. On the cell surface, galactosyltransferase is expressed in spatially restricted, cell type-specific domains, where it functions as a receptor for extracellular oligosaccharide ligands during selected cellular interactions. For example, galactosyltransferase is found on the leading and trailing edges of migrating cells, where it facilitates lamellipodia formation and cell spreading by binding to specific N-linked oligosaccharides within laminin. Although the ability of galactosyltransferase to serve as a laminin receptor is well documented, it is unclear whether it functions solely in a lectin-like capacity to bind laminin glycoside ligands or uses its intrinsic catalytic activity to release itself from and modify its oligosaccharide substrate. In this study, whether cell surface galactosyltransferase spontaneously galactosylates laminin matrices was studied during cell migration using endogenous galactose donors. Migrating cells deposit covalently bound galactose residues onto the laminin matrix. The degree of galactosylation by transfer is both laminin- and time-dependent and requires actively migrating, intact cells. Galactosylation is dependent upon galactosyltransferase-mediated cell migration, since prelabeled cells did not deposit galactose when migrating on fibronectin, upon which migration is integrin-dependent and galactosyltransferase-independent. These results raise the possibility that galactosyltransferase functions catalytically during cell migration, either to dissociate from its oligosaccharide ligand and/or to modify the extracellular matrix (Begovac, 1994).

Structural changes in the extracellular matrix are necessary for cell migration during human tissue remodeling and tumor invasion. Specific cleavage of laminin-5 (Ln-5) by matrix metalloprotease-2 (MMP2) induces migration of breast epithelial cells. MMP2 cleaves the Ln-5 gamma2 subunit at residue 587, exposing a putative cryptic promigratory site on Ln-5 that triggers cell motility. This altered form of Ln-5 is found in tumors and in tissues undergoing remodeling, but not in quiescent tissues. Cleavage of Ln-5 by MMP2 and the resulting activation of the Ln-5 cryptic site may provide new targets for modulation of tumor cell invasion and tissue remodeling (Giannelli, 1997).

Laminin mutation

Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are alpha/beta/gamma heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development. The laminin alpha5 chain is required during embryogenesis. The alpha5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose alpha5 retain or acquire other alpha chains. Embryos lacking laminin alpha5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly); failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are alpha5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin alpha chains accumulate in these BLs, but this compensation is apparently functionally inadequate. These results identify new roles for laminins and BLs in diverse developmental processes (Miner, 1999).

Laminin receptors

Continued: see Laminin A Evolutionary homologs part 2/3 | part 3/3


Laminin A: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.