Isolation of NILE Glycoprotein-Related cDNA Probes

The nerve growth factor (NGF)-induced large external (NILE) glycoprotein is an NGF-inducible surface component of PC12 cells that is also widely distributed in the nervous system. It has recently been shown to be indistinguishable from the high-molecular-weight species of the brain antigens L1 and neuron-glia cell adhesion molecule (Ng-CAM) and may have a function in regulating cell adhesion in the developing nervous system. We have used polyclonal anti-NILE antisera to screen a lambda gt11 cDNA library made from NGF-treated PC12 cells. Four molecular probes have been isolated that encode parts of the apoprotein, related proteins, or both. These probes are 1,500, 800, 330, and 300 base pairs long, respectively, and in Northern blots they recognize a family of messages having lengths of approximately 5.9, 3.4, 2.4, and 1.9 kilobases. The two smaller messages are modestly but reproducibly up-regulated by NGF in PC12 cells, as is the NILE glycoprotein; however, only the two larger species would appear to be large enough to encode it. These messages are prominent in brain but not in nonneural tissues, in accordance with the observed levels of the protein. The recombinant phages produce fusion proteins that share specific epitopes with the NILE glycoprotein but not with other proteins. In these experiments, filters coated with recombinant fusion protein were prepared. Antibodies bound to and eluted from these filters specifically immunoprecipitate NILE glycoprotein, but not other proteins, from PC12 cell extracts. Other activities present in the original sera are not specifically retained by recombinant fusion proteins, and Escherichia coli lysates made with nonrecombinant lambda gt11 do not absorb the anti-NILE activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration of immunochemical identity between the nerve growth factor-inducible large external (NILE) glycoprotein and the cell adhesion molecule L1.

The nerve growth factor-inducible large external (NILE) glycoprotein and the neural cell adhesion molecule L1 were shown to be immunochemically identical. Immunoprecipitation with L1 and NILE antibodies of [3H]fucose-labeled material from culture supernatants and detergent extracts of NGF-treated rat PC12 pheochromocytoma cells yielded comigrating bands by SDS-PAGE. NILE antibodies reacted with immunopurified L1 antigen, but not with N-CAM and other L2 epitope-bearing glycoproteins from adult mouse brain. Finally, by sequential immunoprecipitation from detergent extracts of [35S]methionine-labeled early post-natal cerebellar cell cultures or [3H]fucose-labeled NGF-treated PC12 cells, all immunoreactivity for NILE antibody could be removed by pre-clearing with L1 antibody and vice versa.     

Cell-cell interactions modulate the responsiveness of PC12 cells to nerve growth factor

The growth of PC12 cells on a collagen substratum or on monolayers of several non-neuronal cell types was studied by measuring nerve growth factor (NGF)-dependent increases in the expression of a 150 X 10(3) (Mr) neurofilament protein subunit and the membrane glycoprotein Thy-1. Both responses were found to be greatly suppressed in cultures of fibroblasts as compared to the C2 and G8-1 muscle cell lines and the C6 glioma cell line. This suppression was associated with an inhibition of NGF-dependent neuritic outgrowth from PC12 cells grown on fibroblast monolayers. There was no evidence that fibroblasts secrete soluble molecules that directly inhibit these responses or neutralize NGF. In addition, there was no difference in the neurofilament protein response from PC12 cells that had been treated with NGF prior to coculture, and the now primed PC12 cells readily extended axons over fibroblast monolayers. These data demonstrate that cell-cell and/or cell-matrix interactions can modulate biochemical responses to NGF and suggest that responsiveness of neuronal cells to environmental cues is not immutable. Control of the latter may be at the level of expression of receptor molecules for cell-surface- or matrix-associated macromolecules and a similar mechanism operating during development could play a role in growth cone guidance.     

Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions

Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural Features of the Nerve Growth Factor Inducible Large External Glycoprotein of PC12 Pheochromocytoma Cells and Brain

Abstract: We have examined the oligosaccharide structure of a major M_r= 230,000 cell surface glycoprotein from rat PC12 pheochromocytoma cells, and of the immunochemically cross-reactive species present in brain. In response to nerve growth factor (NGF) the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. These morphological changes are accompanied by a 3- to 5-fold increase in the concentration and labeling of this cell surface glycoprotein, which has previously been named the NGF-inducible large external, or NILE, glycoprotein. Tri- and tetraantennary complex oligosaccharides are the predominant carbohydrate units present in the NILE glycoprotein from both brain and PC12 cells, where they represent 77–90% of the biosynthetically labeled oligosaccharides. Most of these are not substituted by fucose on the core N -acetylglucosamine which is linked to asparagine, and are accompanied by smaller proportions of biantennary and high-mannose oligosaccharides. Sequential lectin-agarose affinity chromatography employing concanavalin A, lentil lectin, and the leukoagglutinating lectin of Phaseolus vulgaris , together with neuraminidase treatment of the fractionated glycopeptides, demonstrated a moderate degree of microheterogeneity among the predominant tri- and tetraantennary oligosaccharide units with respect to the presence of core fucose, outer galactose and sialic acid residues, and the substitution positions on the α-linked mannose residues. NGF treatment of the PC12 cells had no significant effect on the oligosaccharide structure of the NILE glycoprotein. The greater molecular size of the PC12 cell NILE glycoprotein as compared to the immunochemically cross-reactive species present in brain (M_r= 205,000) is apparently due to the greater size of the PC12 cell tri- and tetraantennary complex oligosaccharides.     

Structure of a new nervous system glycoprotein, Nr-CAM, and its relationship to subgroups of neural cell adhesion molecules

We have identified and characterized a new glycoprotein in the chicken nervous system using immunological and molecular biological methods and we have examined its tissue distribution. Analysis revealed that this protein is very similar in structure to the chicken neuron-glia cell adhesion molecule, Ng-CAM, and to mouse L1. cDNA clones encompassing the entire coding sequence of this Ng-CAM related molecule, called Nr-CAM, have been isolated and sequenced. A glycoprotein containing one major component of Mr 145,000 on SDS-PAGE was purified from brain by lentil lectin affinity chromatography and FPLC, and its amino-terminal sequence was identical to that predicted from the Nr-CAM cDNA. The complete cDNA sequence encodes six Ig-like domains, five fibronectin type III repeats, a predicted transmembrane domain, and a short cytoplasmic domain. On Northern blots, nucleic acid probes for Nr-CAM recognized one major RNA species of approximately 7 kb and much lesser amounts of larger RNAs. Most of the same probes hybridized to single bands on genomic Southern blots, suggesting that Nr-CAM is encoded by a single gene that may be alternatively processed to yield several mRNAs. In support of this notion, two Nr-CAM cDNA clones had a 57-bp sequence located between the second and third Ig-like domains that was not found in two other Nr-CAM cDNA clones, and two other clones were isolated that lacked the 279-bp segment encoding the fifth fibronectin-like type III repeat. Antibodies against the purified protein and synthetic peptides in Nr-CAM both recognized a predominant Mr 145,000 species and a much less prevalent species of Mr 170,000 in neural tissues. Levels of Nr-CAM expression increased in the brain until approximately embryonic day (E) 12, followed by slightly lower levels of expression at E18 and after hatching. Immunofluorescent staining with anti-Nr-CAM antibodies showed that most neurons in the retina were positive at E7 and the pattern of expression became restricted to several layers on neuronal cell bodies and fibers during development. Anti-Nr-CAM antibodies labeled specifically cell surfaces on neurons in culture. Although the structure of Nr-CAM resembles that of chicken Ng-CAM and mouse L1, the identity with each of these neural CAMs does not exceed 40%. The differences indicate that Nr-CAM is distinct from Ng-CAM and L1, but there are sufficient similarities to suggest that all of these molecules are members of a subgroup of neural CAMs in the N-CAM superfamily.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased Intracellular Cyclic AMP Differentially Modulates Nerve Growth Factor Induction of Three Neuronal Recognition Molecules Involved in Neurite Outgrowth

The relative expression of the immunoglobulin superfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.     

Poly(N-acetyllactosaminyl) oligosaccharides in glycoproteins of PC12 pheochromocytoma cells and sympathetic neurons

Endo-beta-galactosidase treatment of glycopeptides derived from the trypsinate and membranes of PC12 pheochromocytoma cells and cultured sympathetic neurons demonstrated the presence of poly(N-acetyllactosaminyl) units on tri- and tetraantennary oligosaccharides, some of which have a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Nerve growth factor induced differentiation of the PC12 cells led to a small but significant decrease in the proportion of these oligosaccharides. Poly(N-acetyllactosaminyl) oligosaccharides were also identified in a major 230 000-Da cell-surface glycoprotein (the nerve growth factor inducible large external, or NILE, glycoprotein) of PC12 cells and appear to account for much or all of the difference in size between this glycoprotein as compared to the immunochemically cross-reactive 205 000-Da species present in postnatal brain. Glycoproteins containing poly(N-acetyllactosaminyl) oligosaccharides were selectively labeled by treatment of PC12 cells with endo-beta-galactosidase to expose N-acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP-[14C]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed the presence of a number of distinct PC12 cell glycoproteins that contain these oligosaccharides and have apparent molecular weights in the range of 25 000-250 000. Treatment of PC12 cells with nerve growth factor (NGF) altered the relative labeling of several of the glycoprotein bands, with a time course similar to the effects of NGF on neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gicerin/CD146 is involved in neurite extension of NGF-treated PC12 cells

Gicerin/CD146 is a cell adhesion molecule, which belongs to the immunoglobulin (Ig) superfamily. We have reported that it has a homophilic binding activity, which participates in the neurite extension from embryonic neurons. To elucidate how gicerin is involved in the neurite extension mechanism, we employed PC12 cells, which expresses gicerin/CD146. PC12 cells extend longer neurites by nerve growth factor (NGF) on gicerin substrate than on without gicerin substrate, which indicates that gicerin participates in neurite extension by NGF. We also found that the expression of gicerin in PC12 cells is induced by NGF. Over-expression of gicerin also promotes neurite extension by gicerin-gicerin homophilic interaction. These findings suggested that increase of gicerin expression by NGF promotes the gicerin-gicerin homophilic interaction resulting in the neurite extension.     

Probable Identity of NILE Glycoprotein and the High-Molecular-Weight Component of L1 Antigen

We report here that anti-L1 antiserum, raised against material from embryonic brain, and anti-NILE antiserum, raised against purified NILE (nerve growth factor-inducible large external) glycoprotein of PC12 cells, immunoprecipitate from PC12 cells material of the same apparent molecular weight (230 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, each of these immune reagents has the capacity to clear from a PC12 cell extract all of the 230-kilodalton antigen recognized by the other antiserum. Finally, in immunohistochemical staining of developing cerebellum the two antisera exhibit very similar staining patterns. We suggest that the NILE glycoprotein and the high-molecular-weight component of L1 antigen are closely related molecules, and probably the same.     

Initial appearance and regional distribution of the neuron-glia cell adhesion molecule in the chick embryo

This study represents a global survey of the times of the first appearance of the neuron-glia cell adhesion molecule (Ng-CAM) in various regions and on particular cells of the chick embryonic nervous system. Ng-CAM, originally characterized by means of an in vitro binding assay between glial cells and brain membrane vesicles, first appears in development at the surface of early postmitotic neurons. By 3 d in the chick embryo, the first neurons detected by antibodies to Ng-CAM are located in the ventral neural tube; these precursors of motor neurons emit well-stained fibers to the periphery. To identify locations of appearance of Ng-CAM in the peripheral nervous system (PNS), we used a monoclonal antibody called NC-1 that is specific for neural crest cells in early embryos to show the presence of numerous crest cells in the neuritic outgrowth from the neural tube; neither these crest cells nor those in ganglion rudiments bound anti-Ng-CAM antibodies. The earliest neurons in the PNS stained by anti-Ng-CAM appeared by 4 d of development in the cranial ganglia. At later stages and progressively, all the neurons and neurities of the PNS were found to contain Ng-CAM both in vitro and in vivo. Many central nervous system (CNS) neurons also showed Ng-CAM at these later stages, but in the CNS, the molecule was mostly associated with neuronal processes (mainly axons) rather than with cell bodies; this regional distribution at the neuronal cell surface is an example of polarity modulation. In contrast to the neural cell adhesion molecule and the liver cell adhesion molecule, both of which are found very early in derivatives of more than one germ layer, Ng-CAM is expressed only on neurons of the CNS and the PNS during the later epoch of development concerned with neural histogenesis. Ng-CAM is thus a specific differentiation product of neuroectoderm. Ng-CAM was found on developing neurons at approximately the same time that neurofilaments first appear, times at which glial cells are still undergoing differentiation from neuroepithelial precursors. The present findings and those of previous studies suggest that together the neural cell adhesion molecule and Ng-CAM mediate specific cellular interactions during the formation of neuronal networks by means of modulation events that govern their prevalence and polarity on neuronal cell surfaces.     

Neuron-glia cell adhesion molecule interacts with neurons and astroglia via different binding mechanisms

The neuron-glia cell adhesion molecule (Ng-CAM) is present in the central nervous system on postmitotic neurons and in the periphery on neurons and Schwann cells. It has been implicated in binding between neurons and between neurons and glia. To understand the molecular mechanisms of Ng-CAM binding, we analyzed the aggregation of chick Ng-CAM either immobilized on 0.5-micron beads (Covaspheres) or reconstituted into liposomes. The results were correlated with the binding of these particles to different types of cells as well as with cell-cell binding itself. Both Ng-CAM-Covaspheres and Ng-CAM liposomes individually self-aggregated, and antibodies against Ng-CAM strongly inhibited their aggregation; the rate of aggregation increased approximately with the square of the concentration of the beads or the liposomes. Much higher rates of aggregation were observed when the ratio of Ng-CAM to lipid in the liposome was increased. Radioiodinated Ng-CAM on Covaspheres and in liposomes bound both to neurons and to glial cells and in each case antibodies against Ng-CAM inhibited 50-90% of the binding. Control preparations of fibroblasts and meningeal cells did not exhibit significant binding. Adhesion between neurons and glia within and across species (chick and mouse) was explored in cellular assays after defining markers for each cell type, and optimal conditions of shear, temperature, and cell density. As previously noted using chick cells (Grumet, M., S. Hoffman, C.-M. Chuong, and G. M. Edelman. 1984 Proc. Natl. Acad. Sci. USA. 81:7989-7993), anti-Ng-CAM antibodies inhibited neuron-neuron and neuron-glia binding. In cross-species adhesion assays, binding of chick neurons to mouse astroglia and binding of mouse neurons to chick astroglia were both inhibited by anti-Ng-CAM antibodies. To identify whether the cellular ligands for Ng-CAM differed for neuron-neuron and neuron-glia binding, cells were preincubated with specific antibodies, the antibodies were removed by washing, and Ng-CAM-Covasphere binding was measured. Preincubation of neurons with anti-Ng-CAM antibodies inhibited Ng-CAM-Covasphere binding but similar preincubation of astroglial cells did not inhibit binding. In contrast, preincubation of astroglia with anti-astroglial cell antibodies inhibited binding to these cells but preincubation of neurons with these antibodies had no effect. Together with the data on Covaspheres and liposome aggregation, these findings suggested that Ng-CAM-Covaspheres bound to Ng-CAM on neurons but bound to different molecules on astroglia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Release of the NILE and Other Glycoproteins from Cultured PC 12 Rat Pheochromocytoma Cells and Sympathetic Neurons

Studies were carried out on the glycoproteins (GPs) released by cultured rat sympathetic neurons and by cultured PC12 rat pheochromocytoma cells with and without nerve growth factor (NGF) treatment. Cultures were prelabeled with [3H]fucose and then incubated for 4-8 h in fresh unlabeled medium. The material released into the medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The patterns of labeled material released by all three types of cultures were similar. One of the major components released was of apparent Mr less than or equal to 230,000. Another major component of apparent Mr = 55,000 as well as minor components of apparent Mr less than or equal to 180,000, 140,000, 118,000, and 105,000 were also detected. An additional peptide of apparent Mr less than or equal to 210,000 was released only by the sympathetic neurons. The soluble released Mr less than or equal to 230,000 component appeared to be derived from a previously characterized neuronal integral membrane GP referred to as the NILE (NGF-inducible large external) GP. Evidence for this included recognition of the released component by a monospecific antiserum prepared against membrane-derived NILE GP. At least several of the other released GPs appeared to be derived from membrane-bound components with which they share immuno-crossreactivity. Since the soluble NILE and other released GPs had somewhat faster mobilities on SDS-polyacrylamide gels than their apparent membrane-bound correspondents, release could either be due to, or accompanied by, minor changes in molecular structure.     

Sequential expression and differential function of multiple adhesion molecules during the formation of cerebellar cortical layers

We have correlated the times of appearance of the neural cell adhesion molecule (N-CAM), the neuron-glia cell adhesion molecule (Ng-CAM), and the extracellular matrix protein, cytotactin, during the development of the chicken cerebellar cortex, and have shown that these molecules make different functional contributions to granule cell migration. Immunofluorescent staining showed distinct spatiotemporal expression sequences for each adhesion molecule. N-CAM was present at all times in all layers. However, the large cytoplasmic domain polypeptide of N-CAM was always absent from the external granular layer and was enriched in the molecular layer as development proceeded. Ng-CAM began to be expressed in the premigratory granule cells just before migration and later disappeared from cell bodies but remained on parallel fibers. Cytotactin, which is synthesized by glia and not by neurons, appeared first in a speckled pattern within the external granular layer and later appeared in a continuous pattern along the Bergmann glia; it was also enriched in the molecular layer. After we established their order of appearance, we tested the separate functions of these adhesion molecules in granule cell migration by adding specific antibodies against each molecule to cerebellar explant cultures that had been labeled with tritiated thymidine and then measuring the differential distribution of labeled cells in the forming layers. Anti-N-CAM showed marginal effects. In contrast, anti-Ng-CAM arrested most cells in the external granular layer, while anti-cytotactin arrested most cells in the molecular layer. Time course analyses combined with sequential addition of different antibodies in different orders showed that anti-Ng-CAM had a major effect in the early period (first 36 h in culture) and a lesser effect in the second part of the culture period, while anti-cytotactin had essentially no effect at the earlier time but had major effects at a later period (18-72 h in culture). The two major stages of cerebellar granule cell migration thus appear to be differentially affected by distinct adhesion molecules of different cellular origins, binding mechanisms, and overall distributions. The results indicated that local cell surface modulation of adhesion molecules of different specificities at defined stages and sites is essential to the formation of cerebellar cortical layers.     

Regulation of neural cell adhesion molecule expression on cultured mouse Schwann cells by nerve growth factor.

Schwann cells from early postnatal mouse sciatic nerve were obtained as a homogenous population and shown by indirect immunofluorescence to express the neural cell adhesion molecules L1, N-CAM and J1 and their common carbohydrate epitope L2/HNK-1. L1 and N-CAM are synthesized in molecular forms that are slightly different from those expressed by small cerebellar neurons or astrocytes. As in astrocytes, the J1 antigen is expressed by Schwann cells in multiple forms generally ranging from 160 to 230 kd in the reduced state. J1 is secreted by Schwann cells in a 230-kd mol. wt form. Expression of L1 by Schwann cells can be regulated by nerve growth factor (NGF). L1 expression on the cell surface is increased 1.6-fold in the presence of NGF after 3 days of maintenance in vitro and 3-fold after 16 days. NGF does not change expression of N-CAM. The glia-derived neurite-promoting factor (GdNPF) increases L1 expression by a factor of 1.9 and decreases N-CAM expression by a factor of 0.4 after 3 days in vitro. J1 expression on Schwann cell surfaces remains unchanged in the presence of NGF or GdNPF. Antibodies to NGF abolish the influence of NGF on L1 expression. Addition of NGF antibodies to the Schwann cell cultures without exogenously added NGF decreases L1 expression, indicating that Schwann cells secrete NGF that may influence L1 expression by an autocrine mechanism. Our experiments show for the first time that cell adhesion molecule expression on a non-neuronal cell, the Schwann cell, can be directly regulated by the neurotrophic factor NGF. These observations indicate a considerable degree of 'plasticity' of peripheral glia in regulating cell adhesion molecule expression.     

The small Gtpase ras is involved in growth factor-regulated expression of the alpha1 integrin subunit in PC12 cells

PC12 cells interact with several growth factors (e. g. EGF, FGF, and NGF) via specific tyrosine receptor kinases, resulting in cell proliferation or neuronal differentiation. The small GTPase Ras is known to be involved in downstream signaling of these growth factor receptors. Furthermore, cell-matrix interactions mediated by integrins, as well as integrin-induced signaling, are also involved in growth factor-stimulated signal transduction in PC12 cells. In this study we determined the expression of the alpha1 integrin subunit in response to EGF and NGF in PC12 wild-type (wt) cells, and in PC12 cells overexpressing an inactive H-Ras protein (RasN17). In PC12 wt cells, alpha1 integrin expression is upregulated by EGF and NGF. Cell surface expression of alpha1beta1integrin is also enhanced in growth factor-treated cells. This upregulation leads to increased alpha1beta1-specific adhesion to collagen. In cells expressing the dominant-negative RasN17 variant, alpha1 integrin expression and alpha1beta1-specific adhesion remain unchanged in response to both growth factors.