Characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1), 10 (alpha5beta1gamma1).

Laminins, a family of large heterotrimeric (alphabetagamma) proteins, are major components of basement membranes implicated in a variety of cellular functions. Different commercial laminin preparations isolated from human placenta have been widely used in functional studies but their molecular properties are poorly known. In the present study, we characterized several of these preparations by ELISA, silver staining and Western blotting, in comparison to mouse laminin 1 (alpha1beta1gamma1), and recombinant human laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1) and 10 (alpha5beta1gamma1). The cell migration-promoting activity of different batches was also tested. The placenta laminin preparations differed from one another and consisted of highly fragmented proteins, a mixture of laminin isoforms, and/or contaminating fibronectin. Major functional differences between batches were also observed, reflecting molecular heterogeneity. Previous data obtained in functional studies using these preparations need to be interpreted with caution and may require revision, and future functional studies demand prior molecular characterization of the laminins, particularly their alpha-chain.     

Recombinant human laminin-10 (alpha5beta1gamma1). Production, purification, and migration-promoting activity on vascular endothelial cells

The laminin (LN) family of large heterotrimeric extracellular matrix glycoproteins has multiple functions: LNs take part in the regulation of processes such as cell migration, differentiation, and proliferation, in addition to contributing to the structure of basement membranes. LN-10, composed of alpha5, beta1, and gamma1 chains, is widely distributed in most basement membranes of both epithelia and endothelia. We determined the complete human cDNA sequence for the LN alpha5 chain and produced recombinant human LN-10 (rLN-10) in HEK293 cells by triple transfection of full-length cDNAs encoding the human LN alpha5, beta1, and gamma1 chains. The rLN-10 was purified using affinity chromatography and had an apparent molecular mass of approximately 800 kDa in SDS-PAGE and a native domain structure in rotary shadowing electron microscopy. By using function-blocking monoclonal antibodies, integrin alpha(3)beta(1) was found to be a major mediator of adhesion of HT-1080 and human saphenous vein endothelial cells. Human saphenous vein endothelial cells adhered more strongly to rLN-10 than to LN-1 and LN-8 and showed better migration on rLN-10, compared with several other matrices. Considering the cell adhesive and migration-promoting properties of rLN-10 on endothelial cells, this molecule could be useful in improving the biocompatibility and endothelialization of vascular grafts.     

Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of alpha5-laminin(s) and alpha3beta1 integrin

Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of alpha2-, alpha3-, alpha4- and alpha5-, but not alpha1-, laminins by the tumor vasculature. In functional assays, alpha3 (Lm-332/laminin-5)- and alpha5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. alpha1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas alpha2 (Lm-211/laminin-2)- and alpha4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified alpha3beta1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to alpha3beta1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete alpha2-, alpha4- and alpha5-laminins and that alpha3- and alpha5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify alpha3beta1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin.     

Recombinant laminin-8 (alpha(4)beta(1)gamma(1)). Production, purification,and interactions with integrins

Laminins are a large family of heterotrimeric extracellular matrix glycoproteins that, in addition to having structural roles, take part in the regulation of processes such as cell migration, differentiation, and proliferation. The laminin alpha(4) chain is widely distributed both in adults and during development in tissues such as cardiac, skeletal and smooth muscle fibers, vascular endothelia, lungs, and in peripheral nerves. It can associate with laminin beta(1)/gamma(1) chains to form laminin-8 and with the beta(2)/gamma(1) chains to form laminin-9. Functional studies on these laminins have been hampered by poor availability of the protein in pure and soluble forms. To facilitate studies on laminin-8, recombinant laminin-8 was produced in a mammalian expression system, purified and shown to form native Y-shaped molecules in rotary shadowing electron microscopy. Integrins mediating cell adhesion to laminin-8 were identified using function-blocking mAbs. The integrin specificities were found to differ somewhat from that of laminin-1. Integrin alpha(6)beta(1) was found to be a major mediator of adhesion of HT-1080 and cultured capillary endothelial cells to laminin-8. Considering the expression patterns of laminin-8 and integrin alpha(6)beta(1) it is likely that the former is a ligand for the latter in vivo as well.     

A neuronal cell line (PC12) expresses two beta 1-class integrins-alpha 1 beta 1 and alpha 3 beta 1-that recognize different neurite outgrowth-promoting domains in laminin

Integrins mediate neuronal process outgrowth on components of the ECM. Integrin alpha subunit-specific antibodies have been used to examine the roles of individual beta 1 integrins in attachment and neurite outgrowth by the neuronal cell line, PC12, in response to laminin and collagen. alpha 1 beta 1 and alpha 3 beta 1 were identified as the major beta 1 integrins expressed by PC12 cells. In functional assays, both alpha 1 beta 1 and alpha 3 beta 1 mediated PC12 cell interactions with laminin, whereas alpha 1 beta 1 alone mediated responses to collagen types I and IV. alpha 1 beta 1 and alpha 3 beta 1 were shown to recognize two different neurite-promoting sites in laminin: alpha 1 beta 1 interacted with the cross-region of laminin present in proteolytic fragments E1-4 and E1; alpha 3 beta 1 recognized a site in the long arm contained in laminin fragment E8. Thus, PC12 cells express two beta 1 integrins, which together function in attachment and neurite outgrowth on laminin and collagen. These integrins are candidates for mediating neurite outgrowth of sympathetic and other neurons in response to these ECM components.     

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.     

Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin.

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.     

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed in combination with beta2 or beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Characterization of the ligand-binding specificities of integrin alpha3beta1 and alpha6beta1 using a panel of purified laminin isoforms containing distinct alpha chains

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11 > laminin-5 > laminin-1 > laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.     

Beta 8 integrins mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin.

Integrins are major receptors used by cells to interact with extracellular matrices. In this paper, we identify the first ligands for the beta 8 family of integrins, presenting evidence that integrin heterodimers containing the beta 8 subunit mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin. A polyclonal antibody, anti-beta 8-Ex, was prepared to a bacterial fusion protein expressing an extracellular portion of the chicken beta 8 subunit. In nonreducing conditions, this antibody immunoprecipitated from surface-labeled embryonic dorsal root ganglia neurons a M(r) 100 k protein, the expected M(r) of the beta 8 subunit, and putative alpha subunit(s) of M(r) 120 k. Affinity-purified anti-beta 8-Ex strongly inhibited sensory neurite outgrowth on laminin-1, collagen IV, and fibronectin-coated substrata. Binding sites were identified in a heat-resistant domain in laminin-1 and in the carboxyl terminal, 40-kDa fibronectin fragment. On substrates coated with the carboxyl terminal fragment of fibronectin, antibodies to beta 1 and beta 8 were only partially effective alone, but were completely effective in combination, at inhibiting neurite outgrowth. Results thus indicate that the integrin beta 8 subunit in association with one or more alpha subunits forms an important set of extracellular matrix receptors on sensory neurons.     

Agonist-dependent single channel current and gating in alpha4beta2delta and alpha1beta2gamma2S GABAA receptors

The family of gamma-aminobutyric acid type A receptors (GABA(A)Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABA(A)Rs that are mainly comprised of alpha(1), beta(2), and gamma(2) subunits, whereas tonic inhibition is mediated by extrasynaptic GABA(A)Rs comprised of alpha(4/6), beta(2), and delta subunits. We investigated the activation properties of recombinant alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at alpha(4)beta(2)delta GABA(A)Rs (beta approximately 1600 s(-1)) than at alpha(1)beta(2)gamma(2S) GABA(A)Rs (beta approximately 460 s(-1)), whereas GABA activated alpha(1)beta(2)gamma(2S) GABA(A)Rs more rapidly (beta approximately 1800 s(-1)) than alpha(4)beta(2)delta GABA(A)Rs (beta < 440 s(-1)). Single channel recordings of alpha(1)beta(2)gamma(2S) and alpha(4)beta(2)delta GABA(A)Rs showed that both channels open to a main conductance state of approximately 25 pS at -70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited approximately 36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (P(O) approximately 0.3) at alpha(4)beta(2)delta GABA(A)Rs and at least two "modes" of single channel bursting activity, lasting approximately 100 ms at alpha(1)beta(2)gamma(2S) GABA(A)Rs. The most prevalent bursting mode had a P(O) of approximately 0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" P(O) mode ( approximately 0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs occurred as a single population of bursts (P(O) approximately 0.4-0.5) of moderate duration (approximately 33 ms) that could be described by schemes containing two shut and two open states for both GABA(A)Rs. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance.     

Posttranslational modifications and beta/gamma chain associations of human laminin alpha1 and laminin alpha5 chains: purification of laminin-3 from placenta

Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 laminin alpha5 chain is glycosylated into a M(r) 390,000 form that upon secretion is further processed into a M(r) 380,000 form. Hence, despite the shorter peptide length of alpha1 chain in comparison with the alpha5 chain, secreted alpha1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin alpha1 and laminin alpha5 chains in laminin-1 and laminin-10. In placenta laminin alpha1 chain (M(r) 400,000) and laminin alpha5 chain (M(r) 380, 000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin beta2 chains. Surprisingly, a fraction of the laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing laminin isoforms.     

The alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin

This study was undertaken to determine the roles of individual alpha/beta 1 integrin heterodimers in promoting cellular interactions with the different attachment-promoting domains of laminin (LN). To do this, antibodies to the integrin beta 1 subunit or to specific integrin alpha subunits were tested for effects on cell attachment to LN, to elastase fragments E1-4 and E1, derived from the short arms and core of LN's cruciform structure, and to fragment E8 derived from the long arm of this structure. The human JAR choriocarcinoma cells used in this study attached to LN and to fragments E1 and E8. Attachment to E1-4 required a much higher substrate coating concentration, suggesting that it is a poor substrate for JAR cell attachment. The ability of cells to attach to different LN domains suggested the presence of more than one LN receptor. These multiple LN receptors were shown to be beta 1 integrin heterodimers because antibodies to the integrin beta 1 subunit inhibited attachment of JAR cells to LN and its three fragments. To identify the individual integrin alpha/beta 1 heterodimers that mediate interactions with these LN domains, mAbs specific for individual beta 1 heterodimers in human cells were used to study JAR cell interactions with LN and its fragments. An anti-alpha 6/beta 1-specific mAb, GoH3, virtually eliminated cell attachment to E8 and partially inhibited attachment to E1 and intact LN. Thus the major alpha 6/beta 1 attachment domain is present in fragment E8. An alpha 1/beta 1-specific mAb (S2G3) strongly inhibited cell attachment to collagen IV and partially inhibited JAR attachment to LN fragment E1. Thus, the alpha 1/beta 1 heterodimer is a dual receptor for collagen IV and LN, interacting with LN at a site in fragment E1. In combination, the anti-alpha 1- and anti-alpha 6-specific antibodies completely inhibited JAR cell attachment to LN and fragment E1. Thus, the alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers each function as LN receptors and act together to mediate the interactions of human JAR choriocarcinoma cells with LN.     

Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)     

AS160 phosphorylation is associated with activation of alpha2beta2gamma1- but not alpha2beta2gamma3-AMPK trimeric complex in skeletal muscle during exercise in humans

We investigated time- and intensity-dependent effects of exercise on phosphorylation of Akt substrate of 160 kDa (AS160) in human skeletal muscle. Subjects performed cycle exercise for 90 min (67% VO2 peak, n=8), 20 min (80% VO2 peak, n=11), 2 min (110% of peak work rate, n=9), or 30 s (maximal sprint, n=10). Muscle biopsies were obtained before, during, and after exercise. In trial 1, AS160 phosphorylation increased at 60 min (60%, P=0.06) and further at 90 min of exercise (120%, P<0.05). alpha2beta2gamma3-AMP-activated protein kinase (AMPK) activity increased significantly to a steady-state level after 30 min, whereas alpha2beta2gamma1-AMPK activity increased after 60 min of exercise with a further significant increase after 90 min. alpha2beta2gamma1-AMPK activity and AS160 phosphorylation correlated positively (r2=0.55). In exercise trials 2, 3, and 4, alpha2beta2gamma3-AMPK activity but neither AS160 phosphorylation nor alpha2beta2gamma1-AMPK activity increased. Akt Ser473 phosphorylation was unchanged in all trials, whereas Akt Thr308 phosphorylation increased significantly in trial 3 and 4 only. These results show that AS160 is phosphorylated in a time-dependent manner during moderate-intensity exercise and suggest that alpha2beta2gamma1- but not alpha2beta2gamma3-AMPK may act in a pathway responsible for exercise-induced AS160 phosphorylation. Furthermore, we show that AMPK complexes in skeletal muscle are activated differently depending on exercise intensity and duration.     

The LG1-3 tandem of laminin alpha5 harbors the binding sites of Lutheran/basal cell adhesion molecule and alpha3beta1/alpha6beta1 integrins

The laminin-type globular (LG) domains of laminin alpha chains have been implicated in various cellular interactions that are mediated through receptors such as integrins, alpha-dystroglycan, syndecans, and the Lutheran blood group glycoprotein (Lu). Lu, an Ig superfamily transmembrane receptor specific for laminin alpha5, is also known as basal cell adhesion molecule (B-CAM). Although Lu/B-CAM binds to the LG domain of laminin alpha5, the binding site has not been precisely defined. To better delineate this binding site, we produced a series of recombinant laminin trimers containing modified alpha chains, such that all or part of alpha5LG was replaced with analogous segments of human laminin alpha1LG. In solid phase binding assays using a soluble Lu (Lu-Fc) composed of the Lu extracellular domain and human IgG1 Fc, we found that Lu bound to Mr5G3, a recombinant laminin containing alpha5 domains LN through LG3 fused to human laminin alpha1LG4-5. However, Lu/B-CAM did not bind other recombinant laminins containing alpha5LG3 unless alpha5LG1-2 was also present. A recombinant alpha5LG1-3 tandem lacking the laminin coiled coil (LCC) domain did not reproduce the activity of Lu/B-CAM binding. Therefore, proper structure of the alpha5LG1-3 tandem with the LCC domain was essential for the binding of Lu/B-CAM to laminin alpha5. Our results also suggest that the binding site for Lu/B-CAM on laminin alpha5 may overlap with that of integrins alpha3beta1 and alpha6beta1.     

Blood platelets contain and secrete laminin-8 (alpha4beta1gamma1) and adhere to laminin-8 via alpha6beta1 integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin.     

Sodium channel beta1 subunits promote neurite outgrowth in cerebellar granule neurons

Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel beta1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or beta1(-/-) mice were plated on top of parental, mock-, or beta1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on beta1-transfected monolayers, whereas beta1(-/-) neurons showed no increase compared with control conditions. beta1-mediated neurite extension was mimicked using a soluble beta1 extracellular domain and was blocked by antibodies directed against the beta1 extracellular domain. Immunohistochemical analysis suggests that the beta1 and beta4 subunits, but not beta2 and beta3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for beta1 during neuronal development and are the first demonstration of a functional role for sodium channel beta subunit-mediated cell adhesive interactions.