The I Domain is Essential for Echovirus 1 Interaction with VLA-2

VLA-2, the α2β1 integrin, mediates cell adhesion to collagen and laminin, and is the receptor for the human pathogen echovirus 1. Because of its similarity to domains present in other proteins that interact with collagen, a 191 amino acid region within the α2 subunit (the I domain) has been proposed as a potential site for ligand interactions. Although the α2 subunits of human and murine VLA-2 are 84% identical, human α2 promotes virus binding whereas murine α2 does not. We used murine/human chimeric α2 molecules to identify regions of the human molecule essential for virus binding. Virus bound efficiently to a chimeric protein in which the human I domain was inserted into murine α2, indicating that the human I domain is responsible for specific virus interactions. Monoclonal antibodies that inhibited virus attachment all recognized epitopes within the human I do-main, further suggesting that virus interacts with this portion of the molecule. Similarly, antibodies that prevented VLA-2-mediated cell adhesion to collagen also mapped to the I domain. These results indicate that the I domain plays a role in VLA-2 interactions both with virus and with extracellular matrix ligands.     

Molecular cloning of the rat integrin alpha 1-subunit: a receptor for laminin and collagen

Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.     

Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.     

Determinants of the specificity of rotavirus interactions with the alpha2beta1 integrin

The human α2β1 integrin binds collagen and acts as a cellular receptor for rotaviruses and human echovirus 1. These ligands require the inserted (I) domain within the α2 subunit of α2β1 for binding. Previous studies have identified the binding sites for collagen and echovirus 1 in the α2 I domain. We used CHO cells expressing mutated α2β1 to identify amino acids involved in binding to human and animal rotaviruses. Residues where mutation affected rotavirus binding were located in several exposed loops and adjacent regions of the α2 I domain. Binding by all rotaviruses was eliminated by mutations in the activation-responsive αC-α6 and αF helices. This is a novel feature that distinguishes rotavirus from other α2β1 ligands. Mutation of residues that co-ordinate the metal ion (Ser-153, Thr-221, and Glu-256 in α2 and Asp-130 in β1) and nearby amino acids (Ser-154, Gln-215, and Asp-219) also inhibited rotavirus binding. The importance of most of these residues was greatest for binding by human rotaviruses. These mutations inhibit collagen binding to α2β1 (apart from Glu-256) but do not affect echovirus binding. Overall, residues where mutation affected both rotavirus and collagen recognition are located at one side of the metal ion-dependent adhesion site, whereas those important for collagen alone cluster nearby. Mutations eliminating rotavirus and echovirus binding are distinct, consistent with the respective preference of these viruses for activated or inactive α2β1. In contrast, rotavirus and collagen utilize activated α2β1 and show an overlap in α2β1 residues important for binding.     

Laminin responsiveness is associated with changes in fibroblast morphology,motility, and anchorage-independent growth: Cell system for examining the interaction between laminin and EGF signaling pathways

Laminin can influence the adhesion, differentiation, and motility of motility of several cell types, including epithelial and neural cells. In addition, laminin, which contains an epidermal growth factor (EGF)-like motif, can stimulate DNA synthesis in fibroblasts possessing the EGF receptor, but laminin does not compete for EGF binding. To further investigate laminin action in fibroblasts, and the relationship between laminin and EGF receptor function, we have developed a system wherein cells containing laminin-binding activity were cloned from a mouse fibroblast cell line (B82L-wt) that cannot adhere to laminin but that have been transfected with the wild-type human EGF receptor. Although only the isolated clones can efficiently attach to laminin-coated plates, all the cells can adhere to plastic, fibronectin, and collagen I, and all exhibit comparable levels of cell surface-associated laminin. Ligand-binding assays showed that the cells with laminin attachement activity possess high-affinity EGF binding (Kd ～ 0.4 nM), and all express a similar level of the human EGF receptor. However, when compared to the B82L-wt cells, the cells with laminin-binding activity exhibit altered morphology, anchorage-independent growth, and motility. Specifically, the morphology of the fibroblasts possessing laminin binding activity appears more elongated and they spread more-extensively on plastic plates. Analysis of their growth in soft agar revealed that the clones have a 2-5-fold increase in colony formation in comparison to the B82L-wt cells. The cells possessing laminin attachment ability also exhibit laminin-induced motility, and this movement is directional (chemotaxis) rather than random (chemokinesis), indicating functional laminin receptors and signaling pathways. To examine the specific laminin receptors involved in these effects, the influence of anti-integrin subunit antibodies on cell adhesion and migration was evaluated. These studies showed that an anti-α₆ integrin antibody can completely inhibit the clonal cells' attachment and migration to laminin, and anti-α₆ immunoblots revealed that only the clones express measurable levels of α₆. These data indicate that α₆-containing integrins contribute to the lamininmediated attachment and motility of these clones and that this system may also influence the morphology and anchorage-independent growth of these fibroblasts. In addition, these cells provide a unique system for examining the interaction between EGF and laminin receptor action. © 1995 Wiley-Liss, Inc.     

Alpha 1 beta 1 integrin heterodimer functions as a dual laminin/collagen receptor in neural cells

A monoclonal antibody (3A3) raised against a rat neural cell line (PC12) was shown previously to bind to the surfaces of these cells, inhibiting substratum adhesion. Immunochemical and other data indicated that the heterodimer recognized by 3A3 was a member of the integrin family of adhesive receptors and had a beta 1 subunit. The relationship of the alpha subunit to other integrins was unknown. Here we show that 3A3 recognizes in rat tissues a heterodimer (approximately 185 kDa, approximately 110 kDa; unreduced) that is electrophoretically and immunochemically indistinguishable from the antigen in PC12 cells. Immunoaffinity purification of the heterodimer from neonatal rats and protein microsequencing indicate that the alpha subunit is identical at 11 or 13 N-terminal residues with VLA-1, an integrin on human hematopoietic cells. Monoclonal antibody 3A3 inhibits the attachment of rat astrocytes to laminin or collagen but not to fibronectin or polylysine. These data suggest strongly that the integrin recognized by 3A3 is the rat homologue of VLA-1, i.e., alpha 1 beta 1, and that alpha 1 beta 1 is a dual laminin/collagen receptor.     

Active peptides from the carboxyl-terminal globular domain of laminin α2 and Drosophila α chains

The laminin α1 chain carboxyl-terminal globular domain (G domain) contains multiple biological activities. Recently, we identified five cell binding sequences from the G domain by screening with overlapping 12-mer peptides encompassing the entire domain. The structures of these five sequences in the α1 chain are conserved in the corresponding regions of the different laminin α chains. Here we characterize the adhesion activities of the corresponding peptide segments from both the mouse laminin α2 chain and Drosophila laminin α chain using peptide-coated plastic plates and peptide-conjugated Sepharose beads. Using several cell lines, the laminin α2 chain peptides showed cell attachment and/or spreading activities with cell type specificities. Cell spreading on MG-10 was inhibited by integrin antibodies. Four of the Drosophila laminin peptides showed cell attachment activities. These results suggest that biologically active regions in the G domain are conserved in the laminin α1 and α2 chains, and that these regions in laminin play an important role in cell surface receptor interactions.     

Negative Cooperativity between α3β1and α2β1Integrins in Human Mammary Carcinoma MDA MB 231 Cells

The α₃β₁integrin has been implicated as a receptor for several matrix components, including collagen, fibronectin, and laminins. The function of α₃β₁seems to be very versatile involving cell adhesion to or migration on ECM, establishment of cell–cell contacts in aggregates, as well as linkage to intracellular tyrosine phosphorylation cascades. Here we report a strong induction of attachment of α₃β₁integrin expressing human breast carcinoma cell line MDA MB 231 to matrix proteins by two α₃integrin subunit function-blocking monoclonal antibodies (P1B5 and ASC-1). In contrast, stimulation of adhesion to ECM by inhibitory α₃integrin-specific antibodies was not observed in the α₃β₁integrin-expressing nonmalignant human mammary epithelial cell line MCF-10A or the human breast carcinoma cell line MDA MB 468 that expressed relatively low amounts of α₃β₁integrin at the cell surface. This increase was specific for collagens and not observed on fibronectin or laminin. Physiological concentrations of bivalent cations were not required. MAb P1B5 did not induce homotypic aggregation of MDA MB 231 cells. The P1B5-induced increase in cell attachment to collagens could be prevented but not reduced below control levels by blocking mAb to the α₂integrin subunit. Function blocking anti-α₅integrin subunit mAb was without effect while anti-β₁-mAb completely abolished adhesion. Our data indicate that negative cooperativity between integrins results in transdominant inhibition of α₂β₁function by α₃β₁in human MDA MB 231 but not MDA MB 468 tumor cells or nonmalignant MCF-10A cells.     

Echovirus 1 interaction with the isolated VLA-2 I domain.

The isolated I domain of the integrin VLA-2, produced as a bacterial fusion protein, specifically bound echovirus 1 and prevented virus attachment to cells. These results demonstrate that the receptor structures critical for virus attachment are contained solely within the VLA-2 I domain and that soluble receptor fragments are capable of preventing infection.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

Substrate adhesion of rat hepatocytes: Mechanism of attachment to collagen substrates

Attachment of rat hepatocytes to collagen, which occurs without the aid of fibronectin, was found to be a time-dependent reaction characterized by an initial lag phase of 10–20 min before stable attachment bonds began to form. Increasing the density of molecules in the collagen substrates enhanced the rate of cell attachment. The hepatocytes attached essentially equally well to all the collagen types tested (types I, II, III, IV and V). The initial rate of cell attachment was more rapid to native collagen than to denatured collagen or α1(I) chains, apparently indicating different affinities of the cells for these substrates. However, if cells were incubated for 60 min or more, efficient attachment occurred to the α1(I) chain and to all cyanogen-bromide-treated peptides tested (α1-CB2, α1-CB3, α1-CB4, α1-CB5, α1-CB6A, α1-CB7, α1-CB8, α2-CB2, α2-CB3 and α2-CB4) but not to the aminopropeptide of type I procollagen. A low but significant degree of attachment also took place to substrates made of synthetic peptides with the collagen-like structures (Gly-Ala-Pro)_n, (Gly-Pro-Pro)_n and (Gly-Pro-Hyp)_n, whereas no attachment was observed to polyproline. We suggest that the cell-binding sites in collagen have a simple structure and occur in multiple copies along the collagen molecule. Addition of collagen in solution inhibited intial cell attachment, an effect that persisted longer on substrates made of α1(I) chain than on denatured collagen. The collected data are interpreted in terms of a model for cell-to-collagen adhesion where the formation of stable attachment bonds requires the binding of several low-affinity receptors, clustered at the site of adhesion, to collagen molecules in the substrate.     

T cell receptor-dependent, antigen-specific stimulation of a murine T cell clone induces a transient, VLA protein-mediated binding to extracellular matrix

Upon Ag stimulation, an arsonate-specific murine T cell clone exhibited a rapid but transient increase in cell adhesion to collagen, fibronectin, and laminin. This increase in cell adhesion was not observed when a mutant T cell clone lacking TCR expression was utilized. However, upon stimulation by phorbol esters, both parent and mutant T cell clones exhibited a similar transient increase in adhesion to the three matrix proteins. The observed cell adhesion was extensively inhibited by antibodies to the integrin beta 1 subunit, indicating the involvement of VLA proteins. Despite changes in the adhesive properties, there was essentially no difference in the expression of VLA-1, -3, -4, -5, and -6 between resting and stimulated T cells. Together these results suggest that Ag stimulation transmits signals via the TCR complex resulting in a rapid, but transient, up-regulation of matrix protein binding by VLA proteins already present at the cell surface. Because the appropriate reagents that recognize individual mouse VLA proteins were not available, we used the human T cell line Jurkat to demonstrate that T cell binding to collagen, laminin, and fibronectin is mediated largely by VLA-2, VLA-6, and a combination of VLA-5 and VLA-4, respectively.     

INTEGRIN SYNTHESIS AND UTILIZATION IN CULTURED HUMAN OSTEOBLASTS

This study describes the adhesion of human osteoblasts, culturedin vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77–100%, in 2h and at 55nm substrata concentration, and it was accompained by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the β1 integrin and antibodies to the α5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against α2, α3, α5, αv, β1 andβ3 integrins we detected synthesis of α3β1, α5β1, αvβ3, and an αvβ1-like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus α4, α1 and β5 subunits. In cells adhering in the presence of serum we showed organization of β3 and αv integrins in focal contacts. In cells adhering to fibronectin α5 and β1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.     

Extracellular matrix receptors, ECMRII and ECMRI, for collagen and fibronectin correspond to VLA-2 and VLA-3 in the VLA family of heterodimers

The Very Late Activation Antigen (VLA) proteins are a family of five related heterodimers, which also are part of the integrin superfamily of cell adhesion molecules. Except for the identification of VLA-5 as a fibronectin receptor structure, the functions of the VLA proteins have remained unclarified. In this paper, immunoprecipitation experiments with both anti-alpha and anti-beta subunit antibodies showed that the previously identified cell adhesion receptor for collagen, extracellular matrix receptor II (ECMRII), is equivalent to VLA-2. At the same time a previously described multispecific cell adhesion receptor for collagen, fibronectin, and laminin (ECMRI) has been shown to be identical to VLA-3. Although the mAb 12F1 and P1H5 both recognized VLA-2 (ECMRII), they appeared to define distinct epitopes on the alpha 2 subunit. On the other hand, the mAb P1B5 and J143 recognized the alpha 3 subunit of VLA-3 (ECMRI) at or near the same site. Consistent with the collagen receptor functions of VLA-2 (ECMRII) and VLA-3 (ECMRI), anti-VLA beta antiserum blocked cell attachment to collagen.     

Specific overproduction of very late antigen 1 integrin in two human neuroblastoma cell lines selected for resistance to detachment by an Arg-Gly-Asp-containing synthetic peptide

Very late antigen (VLA) 1 is a member of the family of integral plasma-membrane glycoproteins known as integrins. It is a heterodimer composed of an alpha subunit of Mr 200,000, noncovalently associated with a beta subunit of Mr 110,000 which is shared by other VLA molecules (VLA-2-5). Unlike most of the other VLA proteins which have been shown to be receptors for various extracellular matrix proteins, the ligand for VLA-1 is unknown. Utilizing polyclonal antisera against the human fibronectin receptor as well as alpha subunit-specific monoclonal antibodies and cDNA probes, we have been able to demonstrate that in two human neuroblastoma cell lines, IMR-32 and SK-N-SH, the common beta subunit is associated with alpha 1, alpha 2, alpha 3, and alpha 5 subunits. By culturing these two cell lines in the presence of a synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, which contains the Arg-Gly-Asp cell attachment promotion tripeptide, we have isolated variant cell lines resistant to the detachment effects of this peptide. Peptide-resistant SK-N-SH and IMR-32 neuroblastoma cells exhibit weaker attachment to type I collagen and laminin, but a similar level of attachment to fibronectin as compared to the parental cells. Although the peptide-resistant variant cell lines proliferate at a rate similar to that of the parental cell lines, they stably overproduce (up to 20-fold) the alpha 1 subunit (VLA-1) specifically; and in the IMR-32 variant cells, the common beta 1 subunit is also overproduced. The level of expression of alpha 2 and alpha 3 subunits, however, is considerably reduced and that of the alpha 5 subunit is unchanged relative to the parental cells. These data suggest that the expression of integrin alpha subunits can be regulated differentially and independently of the beta subunit and that the VLA-1 heterodimer has an important function in mediating Arg-Gly-Asp-dependent cell adhesion or other phenotypic properties in human neuroblastoma cells.     

Variants of the α6 β1 Laminin Receptor in Early Murine Development: Distribution,Molecular Cloning and Chromosomal Localization of the Mouse Integrin α6 Subunit

Laminin (A:B1:B2) is a major component of the first basement membrane to appear in the developing mouse embryo. Its effects on morphogenesis and differentiation are mediated by interaction with cell surface receptors that are members of the integrin family. We have studied the expression of the α₆ subunit of murine α₆β₁ and its ligand, laminin, in preimplantation mouse embryos, embryo outgrowths and in embryonic stem (ES) cells and embryonal carcinoma (EC) cells. The α₆ subunit is present in the oocyte and throughout preimplantation development. Laminin A chain appears later than α₆ and has a more restricted distribution until the late blastocyst stage. α₆β₁ is strongly expressed in ES and EC cells; the levels of mRNA expression are not altered by differentiation. Molecular cloning of cDNA for the murine integrin α₆ subunit from a mammary gland γt11 library showed, as in man, an open reading frame encoding two variants of α₆, α₆ and α_6B. The identity of the α₆ amino acid sequence to that in man and chicken is 93% and 73%, respectively. The gene for murine α₆ was mapped to chromosome 2. While undifferentiated ES and EC cells express only α_6B, α_6A is co-expressed in ES cells after differentiation is induced by retinoic acid. α_6B is also the only variant expressed in blastocyst stage embryos, but when blastocysts have grown out in culture both α_6A and α_6B are expressed reflecting the results in the cell lines. We suggest that the deposition of laminin in the embryo is a receptor-mediated process and that the shift in the expression of the variants, as the inner cell mass forms its first differentiated progeny, reflects a change in functional properties.     

Drosophila laminin A chain sequence, interspecies comparison, and domain structure of a major carboxyl portion.

Recent studies ascribed some biological actions of cell adhesion and cell outgrowth to the carboxyl-most 1200 amino acids of vertebrate laminin A chains. Here we report a 6.1-kilobase pair nucleotide cDNA sequence encoding 1951 amino acids and the carboxyl end of a Drosophila laminin A chain. It corresponds to the mouse laminin A domains G, I, II, and III, but may represent a different type of laminin A chain. The arrangement of the cysteine-rich repeats of domain III resembles that of B2 chains. However, it has more amino acid identity with a portion of the mouse laminin A chain domain IIIb than with other laminin repeats. Domains I and II are consistent with an interrupted coiled-coil alpha-helical model of the long arm of laminin but are poorly conserved. The G domain contains five subdomains which are individually related to subdomains of vertebrate laminin A chains. The results indicate that laminin G subdomains should be considered individually, rather than merely as parts of a G-globule. A sequence of hydroxyamino acids contributes to a spacer between two of the subdomains. Stretches of hydroxyamino acids may be indicative of junctions between domains of extracellular Drosophila proteins.     

Reduction of Tumorigenicity by α3 Integrin in a Rhabdomyosarcoma Cell Line

The expression levels of integrin adhesion receptors have often been correlated with neoplastic transformation and invasiveness. To investigate more definitively the role of the integrin VLA-3 (α³β₁) in tumor cell behavior, we transfected α³ subunit cDNA into human rhabdomyosarcoma (RD) cells. Transi ectants expressing high levels of α³β₁, on their cell surface displayed an altered morphology and decreased anchorage-dependent growth in vitro. Cells expressing α³ also displayed marked reduction in anchorage-independent growth in soft agar and in their ability to form tumors when injected subcutaneously into athymic nude mice. Thus, VLA-3 can repress the transformed phenotype of rhabdomyosarcoma tumor cells. Similar changes in morphology and growth characteristics were observed in cells expressing a chimeric molecule X3C4 in which the α³ cytoplasmic domain had been exchanged with that of the α⁴ integrin subunit. Therefore, α³ inhibitory effects in RD cells appear not to require specific signalling through the α³ cytoplasmic domain.     

Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis

In a previous study, we described the cloning of the genes encoding the three subunits of Drosophila laminin, a substrate adhesion molecule, and the cDNA sequence of the B1 subunit (Montell and Goodman, 1988). This analysis revealed the similarity of Drosophila laminin with the mouse and human complexes in subunit composition, domain structure, and amino acid sequence. In this paper, we report the deduced amino acid sequence of the B2 subunit. We then describe the expression and tissue distribution of the three subunits of laminin during Drosophila embryogenesis using both in situ hybridization and immunolocalization techniques, with particular emphasis on its expression in and around the developing nervous system.