Neurite Outgrowth-promoting Activity Research Articles

Domain-specific activation of neuronal migration and neurite outgrowth-promoting activities of laminin

The ECM glycoprotein laminin has profound and varied actions on neurons in vitro. Little is known about how laminin's multiple domains and receptor-binding sites interact in determining its overall effects. Here, it is shown that laminin's ability to promote migration of olfactory epithelium neuronal cells maps to distal long arm domain E8 and is mediated by α 6 β 1 integrin. Surprisingly, treatment of laminin with antibodies against its short arms (domains E1′ or P1′ uncovered a new neuronal migration-promoting activity, mediated by a β 1 integrin other than α 6 β 1 Laminin treated with anti-short arm antibodies also promoted β 1 integrin-dependent neurite outgrowth from late embryonic retinal neurons, which are normally unresponsive to laminin. These “antibody-induced” migration and neurite outgrowth activities mapped to laminin's distal long arm, far from the site(s) of antibody binding. Evidence is presented that the induced activities are not actually cryptic in laminin, but are suppressed by an activity that is located in laminin's P1′ domain and that may be lacking in the laminin homolog merosin.

Isolation and Characterization of Glial Cell Lines from Xenopus Neuroepithelium and Retinal Pigment Epithelium

We have isolated several immortal cell lines from Xenopus neuroepithelium and retinal pigment epithelium. These cell lines were initially isolated from primary cultures by serial passaging of proliferating cells, followed by subcloning with limiting dilution techniques. Several morphologically distinct cell lines have been isolated using these procedures. On the basis of immunocytochemical characterization using specific antibodies, we have established that three of these cell lines, the XR1, XRpe1, and XRpe2 cell lines, are glial-like in nature. These cell lines were extensively labeled by antibodies against glial fibrillary acidic protein and vimentin, markers used to identify glial cells. Mono-layers of these cell lines served as useful substrates for axon outgrowth from developing retinal ganglion cells. In addition, analysis of cell-free substrates, prepared by treatment of cell line monolayers with Triton X- 100, revealed that the XR1, XRpe1, and XRpe2 cell lines produce an extracellular matrix (ECM) with potent neurite outgrowth-promoting activity. In contrast, other established retinal and nonretinal Xenopus cell lines were relatively ineffective and did not support axon outgrowth. We propose that neurite outgrowth-promoting activity produced by these cell lines is associated with their ECM and may be glial cell specific. In addition, to further characterize these cell lines, we have recently imaged live cells, using the atomic force microscope (AFM). The use of AFM on living, cultured cells provides a new, high-resolution method for examining dynamic cytoskeletal and morphological events.

Novel neurite growth-inhibitory properties of an astrocyte proteoglycan

Conditioned medium (CM) of primary cultures of GFAP-positive adherent astrocytes from neonatal rat neocortex contained a chondroitin sulphate/dermatan sulphate proteoglycan (CDSPG) that co-eluted with a heparan sulphate proteoglycan (HSPG) by ion-exchange chromatography. The CDSPG was resolved from the HSPG by molecular sieve chromatography, which indicated that the molecular mass of the HSPG was greater than 300 kDa, while that of the CDSPG was approximately 50 kDa. Specific lyase digestion and urea/polyacrylamide gel electrophoresis established the homogeneity of the CDSPG and suggested molecular masses of the core protein and glycosylated protein as 54 kDa and 58 kDa respectively. Virtually all of the poly- d-lysine substrate-bound proteoglycan-associated neurite growth-promoting activity of astrocyte CM was accounted for by the HSPG. On poly- d-lysine the immobilized CDSPG displayed little neurite growth-stimulatory activity relative to the HSPG. However, the CDSPG inhibited the potent growth-promoting activity of the HSPG by displacing it from the poly- d-lysine substrate. Differential cellular regulation of production of growth-modulatory proteins with different binding avidity for the substrate of growth may determine the success of a regenerative axonal response by fully competent neurons.

The axonal recognition molecule F11 is a multifunctional protein: Specific domains mediate interactions with Ng-CAM and restrictin

F11 κ a glycosyl phosphatidylinositol-anchored axonal surface glycoprotein belonging to a neural subgroup of the immunoglobulin superfamily. In this report, we demonstrate that the F11 protein displays three distinguishable activities: binding to the cell recognition molecule Ng-CAM, interaction with the extracellular matrix glycoprotein restrictin (RN), and a neurite outgrowth-promoting activity. By analyzing deletion mutants expressed in transfected COS cells, epitope mapping of monoclonal antibodies, and neurite outgrowth assays, we reveal that these activities can be localized to distinct regions within the F11 protein. The Ng-CAM-binding site resides in the first two immunoglobulin-like domains of F11, whereas the RN-binding site resides in the second or third domain. A neurite outgrowth-promoting activity of F11 characterized by in vitro culture of tectal cells is independent of F11-Ng-CAM and F11-RN binding.

A variant form of laminin is responsible for the neurite outgrowth-promoting activity in conditioned medium from a squamous carcinoma cell line.

We examined the effects of conditioned medium (CM) obtained from a squamous carcinoma cell line, termed SAS, on chick sympathetic neuritic outgrowth. Neurons grown on a substratum coated with CM extended their neurites. Antisera raised against human laminin and mouse EHS laminin immunoprecipitated the neurite outgrowth-promoting factor in CM. The most purified fraction of CM contained a 740-kDa protein which reacted with the anti-laminin sera and was composed of three polypeptides of 330 kDa, 215 kDa and 195 kDa. The 215-kDa and 195-kDa polypeptides, but not the 330-kDa polypeptide were shown to be antigenically related to mouse laminin and human laminin by immunoblotting. The presence of merosin M chain in SAS cells was ruled out by using the method of reverse transcription polymerase chain reaction (RT-PCR). These results suggest that the 740-kDa protein is a laminin variant having a novel heavy chain of 330 kDa and is responsible for neurite outgrowth-promoting activity in CM.

Secretion and biological activities of heparin-binding growth-associated molecule. Neurite outgrowth-promoting and mitogenic actions of the recombinant and tissue-derived protein.

The cDNA for the developmentally regulated, neurite outgrowth-promoting protein HB-GAM (heparin-binding growth-associated molecule) was recently cloned and shown to encode a novel lysine-rich sequence that is homologous with retinoic acid-induced sequences suggested to function in cell differentiation (Merenmies, J., and Rauvala, H. (1990) J. Biol. Chem. 265, 16721-16724). The same sequence was found for the mitogenic and neurite outgrowth-promoting protein pleiotrophin (Li, Y.-S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M., Kodner, C. M., Milbrandt, J., and Deuel, T. F. (1990) Science 250, 1690-1694). In this study, we have constructed a recombinant baculovirus using the cDNA that encodes the putative preprotein of HB-GAM. The putative secretion signal of HB-GAM is cleaved off in the baculovirus expression system, and the recombinant protein is rapidly secreted to the culture medium. Recombinant HB-GAM purified from the culture medium retains the biochemical characteristics and the neurite outgrowth-promoting activity found for the tissue-derived protein. Studies on the neurite outgrowth-promoting activity suggest that HB-GAM functions as an extracellular matrix-associated protein that enhances axonal growth in perinatal cerebral neurons of the rat. Since the same predicted amino acid sequence has been ascribed to a mitogenic protein, mitogenic activities of the recombinant HB-GAM and of tissue-derived HB-GAM fractions were also studied. Recombinant HB-GAM did not display any significant mitogenic activity, suggesting that tissue-derived HB-GAM preparations may contain other heparin-binding mitogenic factors. We identified in brain-derived HB-GAM fractions a 17-kDa protein (p17) that is detached from heparin by a slightly higher salt concentration as compared to HB-GAM. We suggest that p17 is structurally distinct from HB-GAM and responsible for the mitogenic actions of tissue-derived HB-GAM fractions.

The VASE exon downregulates the neurite growth-promoting activity of NCAM 140.

Axonal growth, guidance and synapse formation are controlled by receptors on neuronal growth cones that can recognize positive and inhibitory cues in the local microenvironment. Four well characterized receptor systems are known that recognize the growth-promoting activities associated with the extracellular matrix and the membranes of cells such as astrocytes, muscle cells and Schwann cells; these are the integrins and the homophilically binding cell adhesion molecules neural-cell adhesion molecule (NCAM), N-cadherin and L1 (refs 5-12). Alternative splicing generates 20-30 isoforms of NCAM and these can also be differentially glycosylated. There are two sites where alternative splicing changes the extracellular structure of membrane-bound NCAM and one of these (the MSD1 region) does not obviously affect function. Here we report that the variable alternatively spliced exon (VASE) in immunoglobulin domain 4 downregulates the neurite outgrowth-promoting activity of NCAM. The high level of VASE expression in the adult central as compared with peripheral nervous system could contribute to the poor regenerative capacity of the former.

Domains of neuronal heparan sulphate proteoglycans involved in neurite growth on laminin

A single neuronal cell assay of neurite growth was utilized to determine types and domains of neuronal proteoglycans involved in neurite growth on laminin. Perturbations of biosynthesis and processing, enzymatic digestion with specific lyases, and competition with glycosaminoglycan side chains produced complementary data consistent with a molecular model implicating glycosaminoglycan (GAG) residues of heparan sulphate proteoglycans (HSPGs) in neurite growth. The observations suggest that HSPGs promote neurite growth on laminin by bridging between binding domains for HSPGs on laminin and on the neuronal cell surface, and that the bridge is tethered at both ends by non-covalent interactions between the binding domains and GAG side chains. Sulphation of the GAGs of HSPGs appears to be critical to the tethering and/or neurite growth-promoting activity of neuronal HSPGs.

Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs

Different neuronal populations were used to compare the neurite outgrowth-promoting activities of N-CAM and N-cadherin expressed via gene transfer on the surface of nonneuronal cells. In contrast to a previously reported developmental loss of retinal ganglion cell responsiveness to N-CAM, these cells exhibited an increased and maintained responsiveness to N-cadherin over the same developmental period (E6–E11). N-CAM and N-cadherin responses could be specifically inhibited by their own antibodies, but not by antisera to the β1 integrin family or the L1/G4 glycoprotein. Cerebellar neurons showed qualitative differences in the nature of the dose-response curves for transfected N-CAM expression (highly cooperative) versus N-cadherin expression (linear). In addition “subthreshold” levels of N-CAM expression, which do not normally support neurite out-growth, did so when coexpressed with functional levels of N-cadherin. These studies show fundamental differences in neuronal responsiveness to cell adhesion molecules and suggest a more dynamic regulation for N-CAM-dependent neurite outgrowth than for N-cadherin-dependent outgrowth.

The contrasting roles of N-CAM and N-cadherin as neurite outgrowthpromoting molecules

The neural cell adhesion molecule (N-CAM) is a prominent member of the immunoglobulin gene superfamily of recognition molecules. It operates in a calcium-independent manner to promote cell-cell adhesion. Alternative splicing of a single gene generates more than twenty N-CAM isoforms and these can be further modified by the differential addition of complex N- and O-linked carbohydrates. In contrast, N-cadherin is a major calcium-dependent adhesion molecule in the brain; it is not a member of the immunoglobulin gene superfamily and, as far as we know, exists as a single gene product with no evidence of differential post-translational modification. Both molecules are believed to operate through a homophilic binding mechanism and both are expressed at key developmental times in a number of tissues including the brain. Antibody perturbation experiments suggest that both of the above cell adhesion molecules (CAMs) can support neurite outgrowth over complex cellular substrata such as astrocytes and Schwann cells. In the present review we discuss the use of a molecular genetic approach to study the neurite outgrowth-promoting activity of these molecules. Using this approach we have found that both CAMs are potent inducers of neurite outgrowth from a variety of neurons. However, whereas a critical value of N-CAM expression is required for increased neurite outgrowth, with small increases above this value having substantial effects, N-cadherin promotes neurite outgrowth in a highly linear manner. In addition, whereas N-CAM promotes chick retinal ganglion cell (RGC) neurite outgrowth at E6 but not E11, N-cadherin does so throughout this developmental period.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Characterization and Developmental Regulation of Two Members of a Novel Human Gene Family of Neurite Outgrowth-Promoting Proteins

This report describes the cloning, expression and characterization of two members of a novel human gene family of proteins, HBNF and MK, which exhibit neurite outgrowth-promoting activity. The HBNF cDNA gene codes for a 168-residue protein which is a precursor for a previously described brain-derived heparin-binding protein of 136 amino acids. The second human gene identified in this study, called MK, codes for a 143-residue protein (including a 22-amino acid signal sequence) which is 46% homologous with HBNF. Complementary DNA constructs coding for the mature HBNF and MK proteins were expressed in bacteria and purified by heparin affinity chromatography. These recombinant proteins exhibited neurite-outgrowth promoting activity, but lacked mitogenic activity. The HBNF gene is expressed in the brain of adult mice and rats, but only minimal expression of MK was observed in this tissue. Different patterns of developmental expression were observed in the embryonic mouse, with MK expression peaking in the brain between days E12 and E14 and diminishing to minimal levels in the adult, while expression of HBNF mRNA was observed to gradually increase during embryogenesis, reaching a maximal level at birth and maintaining this level into adulthood. Expression of these genes was also observed in the human embryonal carcinoma cell line, NT2/D1. Retinoic acid induced the expression of HBNF and MK 6- and 11-fold, respectively, in this cell line. Our studies indicate that HBNF and MK are members of a new family of highly conserved, developmentally regulated genes that may play a role in nervous tissue development and/or maintenance.

Evidence that the B2 chain of laminin is responsible for the neurite outgrowth-promoting activity of astrocyte extracellular matrix

Extracellular matrix (ECM) derived from cerebral cortical astrocytes stimulates neurite outgrowth from pheochromocytoma (PC12) cells in the absence of the classical nerve growth factor (NGF). We have shown here that astrocyte ECM can also stimulate neurite outgrowth from primary cultures of central nervous system (CNS) neurons. Using PC12 cells for a quantitative assay, we also demonstrated that the neurite growth-promoting activity increased as the astrocytes matured in vitro: ECM from older astrocytes (3–12 weeks in vitro) exhibited two-fold more neurite growth-promoting activity than ECM for younger astrocytes (5 days to 2 weeks in vitro). We applied various antibodies to identify the neurite growth-promoting factor of astrocyte ECM and found that anti-laminin inhibited neurite outgrowth by 50%, whereas anti-fibronectin and anti-NGF had no effect. Immunoblots, using laminin chain-specific antibodies, and cDNA hybridization of laminin mRNA demonstrated that cultured astrocytes synthesize only the B2 chain of laminin. This suggests that the B2 chain of laminin suffices to stimulate neurite outgrowth.

The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity

Pathfinding of axons in the developing nervous system is thought to be mediated by glycoproteins expressed on the surface of embryonic axons and growth cones. One molecule suggested to play a role in axonal growth is TAG-1, a 135 kd glycoprotein expressed transiently on the surface of subsets of neurons in the developing mammalian nervous system. We isolated a full-length cDNA clone encoding rat TAG-1. TAG-1 has six immunoglobulin-like domains and four fibronectin type III-like repeats and is structurally similar to other immunoglobulin-like proteins expressed on developing axons. Neurons maintained in vitro on a substrate of TAG-1 extend long neurites, suggesting that this protein plays a role in the initial growth and guidance of axons in vivo. TAG-1 is anchored to the neuronal membrane via a glycosyl phosphatidylinositol linkage and is also released from neurons, suggesting that TAG-1 also functions as a substrate adhesion molecule when released into the extracellular environment.

Axonal regeneration on mature human brain tissue sections in culture.

Fresh frozen sections of the temporal lobe from patients undergoing surgical resection for the control of epilepsy were used as a substrate in tissue culture to test for neurite growth-promoting activity. Robust neurite growth from ganglia of embryonic chicks was observed on gray matter regions of the sections and little growth was observed on white matter regions. These results indicate that the dichotomy between gray and white matter regions of the mature central nervous system in supporting neurite growth is present in human brain tissue and supports the general hypothesis that white matter in the mature central nervous system inhibits neurite regeneration but that gray matter will support neurite growth.

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides

We have identified a synthetic peptide derived from the B2-chain of mouse laminin, Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (p20), which simulates the neurite outgrowth-promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1–5 μM (1–5 μg/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (⪢ 50 μM; ⪢ 50 μg/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth-promoting domain of laminin is located in the α-helical region of the B2-chain, and is active for both central and peripheral neurons.

In vitro evidence for a neurite growth-promoting activity in trembler mouse serum

Basal lamina components, such as heparan sulfate proteoglycan (HSPG) and laminin play an important role in neuritic outgrowth for CNS and PNS neurons in culture. The mutant mouse ‘Trembler’ is characterized by hypomyelinization and production of an excess of basal lamina layers around Schwann cells in peripheral nerves.In order to analyse whether or not the serum of the mutant animals contains neurite outgrowthpromoting factors, we cultured rat spinal cord neurons in the presence of Trembler serum. Under these conditions, the outgrowth of neurites was increased approx, 2 times as compared to control serum. Trembler serum induces neuritic outgrowth characterized both by an increase in number of primary neurites emerging from the nerve cell body as well as by an increase in peripheral branching of neurites.To characterize the factors implicated in this increase we added antibodies directed against HSPG or laminin to the mutant serum. As a result, the increase in neuritic outgrowth was reduced or abolished in both cases.Trembler effect on neurite growth disappeared when the number of the non-neuronal cells was reduced, suggesting that the mutant serum did not act directly on neurons but by the intermediary action of non-neuronal cells.

Sulfated glycosaminoglycans modify growth factor-induced neurite outgrowth in PC12 cells.

Glycosaminoglycans (GAGs), localized on the surfaces of cells and in the basement membrane, modulate the growth and differentiation of many cell types. Recent studies have shown that heparin, a GAG found in mast cells, potentiates the ability of acidic fibroblast growth factor (aFGF) to induce neurite outgrowth in pheochromocytoma (PC12) cells. We examined the effect of a variety of GAGs on aFGF, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The effects observed were dependent upon the specific GAG, the concentration of the GAG, and the growth factor. Heparin potentiated aFGF-induced neurite outgrowth in a concentration-dependent fashion; potentiation increased with increasing heparin concentrations of 0.01-100 micrograms/ml. At concentrations greater than 100 micrograms/ml, heparin potentiation decreased. The maximally active concentration of heparin (100 micrograms/ml) increased the potency of aFGF 102-fold. Increasing concentrations of heparan sulfate, dermatan sulfate, and chondroitin sulfate correlated with increasing aFGF potentiation. The maximally active concentrations of heparan sulfate (100 micrograms/ml), dermatan sulfate (10 mg/ml), and chondroitin sulfate (1 mg/ml) increased the activity of aFGF 11-, 110-, and 11-fold, respectively. Hyaluronic acid did not affect the neurite outgrowth-promoting activity of aFGF. Heparin also altered the activity of bFGF; increasing concentrations of heparin (0.01-1 micrograms/ml) correlated with increased potentiation. At concentrations greater than 1 microgram/ml, heparin concentration was inversely correlated with potentiation. Chondroitin sulfate only increased the percentage of neurite-bearing cells at concentrations greater than 10 micrograms/ml. Maximally active concentrations of heparin (1 microgram/ml) and chondroitin sulfate (1 mg/ml) increased the potency of bFGF 5-fold. The highest concentration of heparan sulfate studied (1 mg/ml) inhibited the activity of bFGF. Dermatan sulfate and hyaluronic acid (0.01-1000 micrograms/ml) had no effect on bFGF activity. Heparan sulfate and chondroitin sulfate showed concentration-dependent potentiation of NGF; maximally active concentrations of heparan sulfate (100 micrograms/ml) and chondroitin sulfate (1 mg/ml) increased the potency of NGF 3-fold, whereas heparin, dermatan sulfate and hyaluronic acid had no effect. None of the GAGs had any effect on PC12 neurite outgrowth when added alone. The specificity of the activity of the GAGs was verified by selective enzyme degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a peripheral nerve neurite growth-promoting activity by development and use of an in vitro bioassay.

The effective regeneration of severed neuronal axons in the peripheral nerves of adult mammals may be explained by the presence of molecules in situ that promote the effective elongation of neurites. The absence of such molecules in the central nervous system of these animals may underlie the relative inability of axons to regenerate in this tissue after injury. In an effort to identify neurite growth-promoting molecules in tissues that support effective axonal regeneration, we have developed an in vitro bioassay that is sensitive to substrate-bound factors of peripheral nerve that influence the growth of neurites. In this assay, neonatal rat superior cervical ganglion explants are placed on longitudinal cryostat sections of fresh-frozen sciatic nerve, and the regrowing axons are visualized by catecholamine histofluorescence. Axons are found to regenerate effectively over sciatic nerve tissue sections. When ganglia are similarly explanted onto cryostat sections of adult rat central nervous system tissue, however, axonal regeneration is virtually absent. We have begun to identify the molecules in peripheral nerve that promote effective axonal regeneration by examining the effect of antibodies that interfere with the activity of previously described neurite growth-promoting factors. Axonal elongation over sciatic nerve tissue was found to be sensitive to the inhibitory effects of INO (for inhibitor of neurite outgrowth), a monoclonal antibody that recognizes and inhibits a neurite growth-promoting activity from PC-12 cell-conditioned medium. The INO antigen appears to be a molecular complex of laminin and heparan sulfate proteoglycan. In contrast, a rabbit antiserum that recognizes laminin purified from mouse Engelbreth-Holm-Swarm (EHS) sarcoma, stains the Schwann cell basal lamina of peripheral nerve, and inhibits neurite growth over purified laminin substrata has no detectable effect on the rate of axonal regeneration in our assay.

Partial purification and characterization of a neurite outgrowth-promoting factor from chick heart-cell conditioned medium

A factor in heart-cell conditioned medium (HCM), which adsorbs to the polyornithine-coated culture substratum, is known to induce neurite outgrowth from neurons in culture. This factor — termed the substrate conditioning factor (SCF) — was purified from chick HCM by fractionation with ammonium sulfate, DEAE Sepharose, and hydroxylapatite. A quantitative bioassay with dorsal root ganglion neurons in dissociated cell culture was used to determine the neurite outgrowth-promoting activity. More than 10,000-fold purification was achieved in the specific activity. SCF was sensitive to trypsin, and bound to the wheat germ lectin affinity column, indicating that SCF is a glycoprotein. The most purified fraction produced two major bands with apparent molecular weights of 360K and 220–240K on SDS-gel electrophoresis under reducing conditions. They were detected as radioactive bands when polypeptides synthesized by heart cells were metabolically labeled with [ 35S]methionine. These results suggest that the 360K and 220–240K components are required for the neurite outgrowth-promoting activity of SCF.

Determination of the molecular weight of neuronectin, a conditioned medium-derived, substrate-binding neurite-extension factor: comparison with laminin using radiation-inactivation analysis

Neurite outgrowth from a wide variety of peripheral neurons is stimulated and may be directed in culture by a substrate-binding factor(s) derived from medium conditioned over numerous types of cells. This factor, or family of factors, which we shall call neuronectin by reason of its ability to serve as an attachment molecule for neurons, has been studied by target-size analysis using radiation inactivation. The radiation-inactivation method has the unique advantage of providing a means for determining the actual functional size of a biologically active molecule irrespective of its state of purification. By this method, the functional size of the major neurite outgrowth-promoting activity (neuronectin) from mouse heart cell conditioned medium has been found to be 350,000 Da. While neuronectin has not yet been purified, determination of the actual functional size provides a framework within which possible models must fit. Thus, although neurite outgrowth-promoting activity in this system is found to be associated with a complex containing laminin, fibronectin, heparan sulfate proteoglycan, and other extracellular matrix molecules, the total size of the functional molecule or molecular complex serving as the major source of activity is limited to 350,000 Da. Consequently, our results suggest that neuronectin from mouse heart cell-conditioned medium is different from laminin (Mr approximately 900,000), a molecule that also exhibits neurite-promoting activity. In addition to the difference in molecular size, neuronectin and laminin differ in that laminin, unlike neuronectin, gives rise to toxic or inhibitory products when exposed to high-energy radiation.