Dystroglycan binding to laminin alpha1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro.

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.

Regulation of neurite outgrowth by integrin activation.

During late-embryonic development, retinal neurons lose the ability to attach and extend neurites on the extracellular matrix molecule laminin-1 (LN-1), despite the fact that they retain expression of integrin receptors for LN-1. Here we show that the developmental loss of responsiveness to LN-1 can be reversed by treatments that increase the activation state of integrins. Both extracellular application of Mn(2+) (at micromolar concentrations) and viral-mediated neuronal expression of a constitutively active form of the ras-related GTPase R-ras (R-ras(38V)) potently promoted late-embryonic retinal neurite outgrowth on LN-1 substrata. In both cases, outgrowth was mediated by integrin alpha6beta1 and not alpha3beta1, even though these neurons express alpha3beta1 and use it for outgrowth on other laminin isoforms, as well as on LN-1 that has been proteolytically or conformationally activated (Ivins et al., 1998). Mn(2+)-and to a much lesser extent R-ras(38V)-also reversed the developmental loss of retinal neuron responsiveness to type IV collagen, by promoting the function of integrin alpha1beta1. Interestingly, the responses of other late-embryonic CNS neurons to LN-1 were also enhanced by treatments that activate integrin function, but those of peripheral nervous system neurons (dorsal root ganglion neurons) were either not enhanced (embryonic neurons) or only modestly improved (adult neurons). These results suggest that a developmental decline occurs in the activation state of neuronal integrins, particularly among CNS neurons. Such a decline may underlie some of the intrinsic loss of regenerative ability sustained by CNS neurons during development and may be a valid target for therapeutic intervention.

Form and function: the laminin family of heterotrimers.

The laminins are a family of glycoproteins that provide an integral part of the structural scaffolding of basement membranes in almost every animal tissue. Each laminin is a heterotrimer assembled from alpha, beta, and gamma chain subunits, secreted and incorporated into cell-associated extracellular matrices. The laminins can self-assemble, bind to other matrix macromolecules, and have unique and shared cell interactions mediated by integrins, dystroglycan, and other receptors. Through these interactions, laminins critically contribute to cell differentiation, cell shape and movement, maintenance of tissue phenotypes, and promotion of tissue survival. Recent advances in the characterization of genetic disruptions in humans, mice, nematodes and flies have revealed developmental roles for the different laminin subunits in diverse cell types, affecting differentiation from blastocyst formation to the post-natal period. These genetic defects have challenged some of the previous concepts about basement membranes and have shed new light on the diversity and complexity of laminin functions as well as established the molecular basis of several human diseases.

Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain.

Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected. By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.

The laminin alpha2 expressed by dystrophic dy(2J) mice is defective in its ability to form polymers.

Mutations in LAMA2 cause severe congenital muscular dystrophy accompanied by nervous system defects [1]. Mice homozygous for the dy(2J) allele of LAMA2 express a laminin alpha2 subunit that has a deletion in the amino-terminal domain VI, providing an animal model for study of the molecular basis of congenital muscular dystrophy [2] [3]. Domain VI is predicted to be involved in laminin polymerization, along with amino-terminal domains from laminin beta and gamma chains [4]. In a solution-polymerization assay, we found that purified dy(2J) laminin assembled poorly and formed little polymer, in contrast to wild-type muscle laminin. Furthermore, dissolution of the collagen IV network caused dy(2J) laminin to be released into solution, indicating that laminin polymers within the skeletal muscle basement membrane were defective. In addition to loss of polymerization, dy(2J) laminin had a reduced affinity for heparin. Finally, recombinant laminin engineered with the dy(2J) deletion was more sensitive to proteolysis and was readily cleaved near the junction of domains V and VI. Thus, the dy(2J) deletion selectively disrupts polymer formation, reduces affinity for heparin, and destabilizes domain VI. These are the first specific functional defects to be identified in a muscular dystrophy laminin, and it is likely that these defects contribute to the abnormalities seen in dy(2J)/dy(2J) muscle and nerve.

Binding of the renal epithelial cell line LLC-PK1 to laminin is regulated by protein kinase C.

The alpha6beta1 integrin heterodimer has been implicated in the mediation of renal epithelial cell binding to laminin, and it has been suggested that this binding is important for renal morphogenesis and development. Studies of nonrenal cells have suggested that the functional activity of alpha6beta1 integrin is regulated by protein kinase C (PKC) activity. In this study, the binding of a renal epithelial cell line, LLC-PK1, to laminin was characterized and the role of PKC activity in the modulation of binding was investigated. LLC-PK1 cells bound to laminin-coated surfaces in a time- and laminin concentration-dependent manner. Binding was strongly inhibited by anti-beta1 integrin antibodies and by anti-alpha6 integrin antibodies. Antibodies against alpha2 integrin and a3 integrin had little inhibitory effect. Cells bound to both whole laminin and laminin fragment E8, i.e., the fragment to which the alpha6beta1 integrin heterodimer binds. Exposure of cells to PKC activators for as little as 2 h enhanced cell binding to laminin approximately twofold, in a protein synthesis-dependent manner. PKC inhibitors antagonized this effect. PKC-stimulated binding was also inhibited by anti-beta1 integrin and anti-alpha6 integrin antibodies. PKC activation did not alter expression of beta1 integrin subunits at the cell surface after short time periods (2 to 4 h), but expression was increased after longer time periods (24 h). These results indicate that the renal epithelial cell line LLC-PK1 binds to laminin via the alpha6betal integrin heterodimer and binding is enhanced by PKC activation. The PKC-mediated enhancement of binding requires protein synthesis and is mediated in part by activation of surface alpha6beta1 integrin.

Laminin polymerization induces a receptor-cytoskeleton network.

The transition of laminin from a monomeric to a polymerized state is thought to be a crucial step in the development of basement membranes and in the case of skeletal muscle, mutations in laminin can result in severe muscular dystrophies with basement membrane defects. We have evaluated laminin polymer and receptor interactions to determine the requirements for laminin assembly on a cell surface and investigated what cellular responses might be mediated by this transition. We found that on muscle cell surfaces, laminins preferentially polymerize while bound to receptors that included dystroglycan and alpha7beta1 integrin. These receptor interactions are mediated through laminin COOH-terminal domains that are spatially and functionally distinct from NH2-terminal polymer binding sites. This receptor-facilitated self-assembly drives rearrangement of laminin into a cell-associated polygonal network, a process that also requires actin reorganization and tyrosine phosphorylation. As a result, dystroglycan and integrin redistribute into a reciprocal network as do cortical cytoskeleton components vinculin and dystrophin. Cytoskeletal and receptor reorganization is dependent on laminin polymerization and fails in response to receptor occupancy alone (nonpolymerizing laminin). Preferential polymerization of laminin on cell surfaces, and the resulting induction of cortical architecture, is a cooperative process requiring laminin- receptor ligation, receptor-facilitated self-assembly, actin reorganization, and signaling events.

Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium leprae.

alpha-Dystroglycan (alpha-DG) is a component of the dystroglycan complex, which is involved in early development and morphogenesis and in the pathogenesis of muscular dystrophies. Here, alpha-DG was shown to serve as a Schwann cell receptor for Mycobacterium leprae, the causative organism of leprosy. Mycobacterium leprae specifically bound to alpha-DG only in the presence of the G domain of the alpha2 chain of laminin-2. Native alpha-DG competitively inhibited the laminin-2-mediated M. leprae binding to primary Schwann cells. Thus, M. leprae may use linkage between the extracellular matrix and cytoskeleton through laminin-2 and alpha-DG for its interaction with Schwann cells.

Neuronal receptors mediating responses to antibodyactivated laminin-1.

Embryonic retinal neurons lose the ability to extend neurites on laminin-1 (LN-1) with increasing developmental age yet still do so on other laminin isoforms. However, after treatment of LN-1 with antibodies to "short-arm" regions or removal of the short arms proteolytically, LN-1 supports attachment and extension of neurites even by late embryonic retinal neurons. We have mapped a domain for antibody-mediated "activation" of LN-1 to the N-terminal end of the alpha1 chain. Furthermore, we show that the primary receptors used in the retinal neuron response to "activated" LN-1 are integrins alpha3 beta1 and alpha6 beta1; these are the same receptors used by these neurons for outgrowth on other LN isoforms. Interestingly, alpha3 beta1 is preferentially involved in neurite outgrowth, whereas alpha6beta1 preferentially mediates attachment and spreading. However, in cultures from alpha3 integrin-deficient mice, alpha6 beta1 mediates retinal ganglion cell neurite outgrowth and compensates for the absence of alpha3 beta1. Finally, we show that key features of the retinal neuron response to LN-1 also characterize neurons of the hippocampus, thalamus, and cerebral cortex; these include poor response to untreated LN-1, extensive neurite outgrowth on antibody-activated LN-1 or on fragment E8, and dependence of this response on integrin alpha6 beta1 and at least one other long arm-binding beta1 integrin. These data suggest that regulation of LN-1 function via the process of activation could have important consequences for axonal regeneration. Curiously, the data also imply that the mechanism of laminin activation involves enhanced function at sites that cannot be considered cryptic.

The laminin alpha2-chain short arm mediates cell adhesion through both the alpha1beta1 and alpha2beta1 integrins.

Laminin-2, a heterotrimer composed of alpha2, beta1, and gamma1 subunits, is the primary laminin isoform found in muscle and peripheral nerve and is essential for the development and stability of basement membranes in these tissues. Expression of a domain VI-truncated laminin alpha2-chain results in muscle degeneration and peripheral nerve dysmyelination in the dy2J dystrophic mouse. We have expressed amino-terminal domains VI through IVb of the laminin alpha2-chain, as well as its laminin-1 alpha1-chain counterpart, to identify candidate cell-interactive functions of this critical region. Using integrin-specific antibodies, recognition sites for the alpha1beta1 and alpha2beta1 integrins were identified in the short arms of both laminin alpha1- and alpha2-chain isoforms. Comparisons with a beta-alpha chimeric short arm protein possessing beta1-chain domain VI further localized these activities to alpha-chain domain VI. In addition, we found that the laminin alpha2-chain short arm supported neurite outgrowth independent of other laminin-2 subunits. A heparin/heparan sulfate binding activity was also localized to this region of the laminin alpha2 subunit. These data provide the first evidence that domain VI of the laminin alpha2-chain mediates interactions with cell surface receptors and suggest that these integrin and heparin binding sites, alone or in concert, may play an important role in muscle and peripheral nerve function.

Developmental expression of perlecan during murine embryogenesis.

Perlecan is a modular heparan sulfate proteoglycan that is an intrinsic constituent of all basement membranes and extracellular matrices. Because of its strategic position and unique structure, perlecan has been implicated in modulating the activity of various growth factors required for normal development and tissue homeostasis. To gain insights into the potential function of perlecan in vivo, we examined the spatiotemporal distribution of its mRNA and protein core during murine embryogenesis. We utilized a new affinity-purified antibody that recognizes specifically the protein core of perlecan together with an in situ RT-PCR approach to perform a systematic analysis of perlecan expression and deposition during murine ontogeny. Perlecan appeared early (E10.5) in tissues of vasculogenesis including heart, pericardium, and major blood vessels. Its early expression coincided with the development of the cardiovascular system. Subsequently (E11-13), the greatest deposition of perlecan occurred within the developing cartilage, especially the cartilage undergoing endochondral ossification, where it remained elevated throughout all the developmental stages, and up to adulthood. Interestingly, the mRNA levels of perlecan were always higher in all the vascularized tissues, principally within endothelial cells, while chondrocytes displayed relatively low mRNA levels. This suggests a higher biosynthesis and turnover rates in the blood vessels vis-à-vis those of cartilaginous and other mesenchymal tissues. During later stages of development (E13-17.5) perlecan mRNA levels progressively increased and its expression correlated with the onset of tissue differentiation of various parenchymal organs including the developing kidneys, lungs, liver, spleen, and gastrointestinal tract. The central nervous system showed no perlecan expression with the exception of the calvaria and choroid plexus. Collectively, the results indicate that perlecan may play crucial roles not only in vasculogenesis but also in the maturation and maintenance of differentiated tissues, including cartilage.

The alpha chain of laminin-1 is independently secreted and drives secretion of its beta- and gamma-chain partners.

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 were expressed in all combinations, transiently and/or stably, in a near-null background. In the absence of its normal partners, the alpha chain was secreted as intact protein and protein that had been cleaved in the coiled-coil domain. In contrast, the beta and gamma chains, expressed separately or together, remained intracellular with formation of betabeta or betagamma, but not gammagamma, disulfide-linked dimers. Secretion of the beta and gamma chains required 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 revealed 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. We conclude 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.

Mild congenital muscular dystrophy in two patients with an internally deleted laminin alpha2-chain.

Congenital muscular dystrophy (CMD) is a group of clinically and genetically heterogeneous disorders inherited in an autosomal recessive mode. The alpha2-chain of laminin-2 (previously called merosin) has been shown by immunohistochemical and genetic analyses to be implicated in the pathogenesis of the 'classic' form of CMD. In the 'merosin-deficient' subgroup, which represents about half of the cases, more definite evidence of the involvement of the laminin alpha2-chain has recently been reported with the identification of mutations in the gene encoding the alpha2-chain of laminin 2 (LAMA2) in CMD patients. Here we report on two siblings from a consanguineous family expressing an internally deleted laminin alpha2-chain as a result of a splice site mutation in the LAMA2 gene which causes the splicing of exon 25. The predicted protein lacks 63 amino acids in domain IVa which forms a globular structure on the short arm of the alpha2-chain. Interestingly, these patients appear mildly affected compared to others who completely lack this protein. This situation presents a striking analogy with Becker muscular dystrophy, where in-frame deletions in the dystrophin gene result in the expression of a semi-functional protein and lead to a mild phenotype.

Neural targeting of Mycobacterium leprae mediated by the G domain of the laminin-alpha2 chain.

We report that the molecular basis of the neural tropism of Mycobacterium leprae is attributable to the specific binding of M. leprae to the laminin-alpha2 (LN-alpha2) chain on Schwann cell-axon units. Using recombinant fragments of LN-alpha2 (rLN-alpha2), the M. leprae-binding site was localized to the G domain. rLN-alpha2G mediated M. leprae binding to cell lines and to sciatic nerves of dystrophic dy/dy mice lacking LN-alpha2, but expressing laminin receptors. Anti-beta4 integrin antibody attenuated rLN-alpha2G-mediated M. leprae adherence, suggesting that M. leprae interacts with cells by binding to beta4 integrin via an LN-alpha2G bridge. Our results indicate a novel role for the G domain of LN-2 in infection and reveal a model in which a host-derived bridging molecule determines nerve tropism of a pathogen.

Laminin-induced clustering of dystroglycan on embryonic muscle cells: comparison with agrin-induced clustering.

The effect of laminin on the distribution of dystroglycan (DG) and other surface proteins was examined by fluorescent staining in cultures of muscle cells derived from Xenopus embryos. Western blotting confirmed that previously characterized antibodies are reactive in Xenopus. In control cultures, alphaDG, betaDG, and laminin binding sites were distributed as microclusters (<1 microm2 in area) over the entire dorsal surface of the muscle cells. Treatment with laminin induced the formation of macroclusters (1-20 microm2), accompanied by a corresponding decline in the density of the microclusters. With 6 nM laminin, clustering was apparent within 150 min and near maximal within 1 d. Laminin was effective at 30 pM, the lowest concentration tested. The laminin fragment E3, which competes with laminin for binding to alphaDG, inhibited laminin-induced clustering but did not itself cluster DG, thereby indicating that other portions of the laminin molecule in addition to its alphaDG binding domain are required for its clustering activity. Laminin-induced clusters also contained dystrophin, but unlike agrin-induced clusters, they did not contain acetylcholine receptors, utrophin, or phosphotyrosine, and their formation was not inhibited by a tyrosine kinase inhibitor. The results reinforce the notion that unclustered DG is mobile on the surface of embryonic muscle cells and suggest that this mobile DG can be trapped by at least two different sets of molecular interactions. Laminin self binding may be the basis for the laminin-induced clustering.

Localization of heparin binding activity in recombinant laminin G domain.

Basement membrane laminin (laminin-1) is a multidomain glycoprotein that interacts with itself, heparin and cells. The interaction with heparin/heparan sulfate proteglycans is thought to be important for the architectural formation of basement membranes and adhesion to cells. The major heparin binding site has been known to reside in the long arm globular domain (G domain). The G domain is in turn subdivided into five subdomains (G1-G5). In order to localize the heparin binding regions further, recombinant G domains (rG and rG5) were expressed in Sf9 insect cells using baculovirus expression vector. By the limited proteolysis of recombinant G domains followed by either heparin affinity HPLC or overlay with radiolabeled heparin, the relative affinity of each subdomain to heparin was assigned as G1>G2 = G4>G5>G3, such that G1 bound strongly and G3 not at all. Since the activity in G1-G3 is cryptic in intact laminin long arm [Sung, U., O'Rear, J. J. & Yurchenco, P. D. (1993) J. Cell Biol. 123, 1255-1268], the active heparin binding site of G domain appears to be located in G4 and proximal G5.

Self-assembly of laminin isoforms.

The alpha, beta, and gamma subunits of basement membrane laminins can combine into different heterotrimeric molecules with either three full short arms (e.g. laminins-1-4), or molecules containing one (laminins-6-9) or more (laminin-5) short arm truncations. Laminin-1 (alpha1beta1gamma1), self-assembles through a calcium-dependent thermal gelation requiring binding interactions between N-terminal short arm domains, forming a meshwork polymer thought to contribute to basement membrane architecture (Yurchenco, P. D., and Cheng, Y. S. (1993) J. Biol. Chem. 268, 17286-17299). However, it has been unclear whether other isoforms share this property, and if so, which ones. To begin to address this, we evaluated laminin-2 (alpha2beta1gamma1), laminin-4 (alpha2beta2gamma1), laminin-5 (alpha3Abeta3gamma2), and laminin-6 (alpha3Abeta1gamma1). The first two isoforms were found to self-aggregate in a concentration- and temperature-dependent manner and a close self-assembly relationship among laminins-1, -2, and -4 were demonstrated by: (a) polymerization of all three proteins was inhibited by EDTA and laminin-1 short arm fragments, (b) polymerization of laminin-1 was inhibited by fragments of laminins-2 and -4, (c) laminin-2 and, to a lesser degree, laminin-4, even well below their own critical concentration, co-aggregated with laminin-1, evidence for co-polymerization. Laminin-5, on the other hand, neither polymerized nor co-polymerized with laminin-1. Laminin-6 failed to co-aggregate with laminin-1 at all concentrations evaluated, evidence for a lack of a related self-assembly activity. The data support the hypothesis that all three short arms are required for self-assembly and suggest that the short arm domain structure of laminin isoforms affect their architecture-forming properties in basement membranes.

Mapping of network-forming, heparin-binding, and alpha 1 beta 1 integrin-recognition sites within the alpha-chain short arm of laminin-1.

Cell-interactive and architecture-forming functions are associated with the short arms of basement membrane laminin-1. To map and characterize these functions, we expressed recombinant mouse laminin-1 alpha-chain extending from the N terminus through one third of domain IIIb. This dumbbell-shaped glycoprotein (r alpha 1(VI-IVb)'), secreted by mammalian cells, was found to possess three activities. 1) Laminin polymerization was quantitatively inhibited by recombinant protein, supporting an alpha-chain role for a three-short arm interaction model of laminin self-assembly. 2) r alpha 1(VI-IVb)' bound to heparin, and the activity was localized to a subfragment corresponding to domain VI by 125I-heparin blotting. 3) PC12 rat pheochromocytoma cells adhered to, and rapidly extended branching neurites on, r alpha 1(VI-IVb)', with adhesion inhibited by alpha 1 and beta 1 integrin chain-specific antibodies. The ability of anti-laminin antibody to block PC12 cell adhesion to laminin was selectively prevented by absorption with r alpha 1(VI-IVb)' or alpha-chain domain VI fragment. This active integrin-recognition site could furthermore be distinguished from a second cryptic alpha 1 beta 1-binding site exposed by heat treatment of fragment P1', a short arm fragment lacking globules. Thus, a polymer-forming, a heparin-binding, and the active alpha 1 beta 1 integrin-recognition site are all clustered at the end of the alpha-chain short arm, the latter two resident solely in domain VI.