Identification of a cell-surface protein involved in PC12 cell- substratum adhesion and neurite outgrowth on laminin and collagen

Turner, David C.; Flier, Leonard A.; Carbonetto, Salvatore

doi:10.1523/jneurosci.09-09-03287.1989

Cited by 135 publications

(124 citation statements)

References 58 publications

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“…In fact. Kramer and Marks (1989) noted that functionally inhibitory antibodies would be necessary to confirm their results since affinity chromatography of integrins in detergent-containing solutions may not always reflect the function of a receptor in vivo (Wayner & Carter, 1987;Hynes et al, 1989). We conclude that the rat and human α 1 β 1 integrins are receptors that bind directly to sites in laminin and collagen.…”

Section: Resultsmentioning

confidence: 99%

.alpha.1.beta.1 Integrin heterodimer functions as a dual laminin/collagen receptor in neural cells

Tawil

Houde²,

Blacher³

et al. 1990

Biochemistry

106

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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 β 1 subunit. The relationship of the α subunit to other integrins was unknown. Here we show that 3A3 recognizes in rat tissues a heterodimer (~185 kDa, ~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 α 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., α 1 β 1 , and that α 1 β 1 is a dual laminin/collagen receptor.Previous reports described attachment and neurite outgrowth of a neural cell line (PC12) on laminin, collagen, and other adhesive proteins Tomaselli et al., 1987Tomaselli et al., , 1988. These functional studies suggested that PC12 cells possess a dual laminin/collagen receptor . Subsequently, monoclonal antibody (mab) 1 3A3 generated against PC12 cells was found to inhibit adhesion to and neurite outgrowth on laminin and collagen . mab 3A3 immunoprecipitates two proteins of ~ 110 and ~ 185 kDa. First, functional and, later, immunochemical data suggested these proteins were members of the integrin family (Hynes, 1987;Buck & Horwitz, 1987;Ruoslahti & Pierschbacher, 1987) of heterodimeric adhesive receptors. Tomaselli et al. (1987Tomaselli et al. ( , 1988 . While the β subunit of this receptor has been identified as β 1 , the identity of the α subunit has not been determined. Since the α subunits in the β 1 integrin family largely determine the ligand binding specificity of each heterodimer (Hynes, 1987;Ruoslahti & Pierschbacher, 1987), it was important to ascertain whether the α subunit had a homologue among the several integrins identified in other species to which its functional properties could be related.Here we show that the heterodimer in PC12 cells recognized by mab 3A3 is expressed in rat tissues where it is electrophoretically and immunologically indistinguishable from that in PC12 cells. Having this rich source of receptor has permitted purication and microsequencing of the larger subunit. The amino acid sequence shows unambiguously that this is an integrin α subunit identical at 11 of the first 13 N-terminal amino acid residues with the α 1 subunit of human VLA-1, an integrin in human hematopoietic cells (Hemler, 1988). These data, together with those of others (Kramer & Marks, 1989;Ignatius & Reichardt, 1988; discussed below), argue strongly that the α 1 β 1 integrin is a dual laminin/collagen integrin is hematopoi...

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Section: Resultsmentioning

confidence: 99%

.alpha.1.beta.1 Integrin heterodimer functions as a dual laminin/collagen receptor in neural cells

Tawil

Houde²,

Blacher³

et al. 1990

Biochemistry

106

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“…Previous studies have reported that integrins function in cell matrix adhesion in PC12 cells (Tomaselli et al, 1988(Tomaselli et al, , 1990Turner et al, 1989). The cvl@l is the dominant integrin-mediating neurite outgrowth, and the a3P 1 heterodimer is a relatively weak receptor (Tomaselli et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

Characterization of neural cell adhesion sites: point contacts are the sites of interaction between integrins and the cytoskeleton in PC12 cells

1994

Self Cite

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As developing or regenerating neurons grow, their axons seek cues in the extracellular matrix that are recognized by integrin receptors. To understand the regulation and structure of neural integrin complexes, we have examined the association of two functionally important integrins, alpha 1 beta 1 and alpha 3 beta 1, within PC12 cells. Detergent-resistant cytoskeletal ghosts were prepared from PC12 cells and examined by immunoblotting. In cells maintained in suspension the alpha 1, alpha 3, and beta 1 integrin subunits were solubilized by Triton X-100 detergent. In contrast, when cells were grown on collagen or laminin about 50% of the alpha 1 and beta 1 subunits were retained with the cytoskeleton, but alpha 3 remained soluble. Confocal immunofluorescence microscopy of whole cells demonstrated that all three integrin subunits were expressed in a punctate pattern on the cell surface in point contacts. Point contacts were also found to be the predominant adhesion structure of dorsal root ganglion neurons. After detergent extraction of PC12 cells, the point contacts remained only at the cell-substrate interface. Vinculin, which is found consistently in focal contacts on non-neural cells, showed only a partial colocalization with the point contacts, being expressed mainly at the tips of filopodia and the periphery of cell bodies. Talin showed no obvious codistribution with beta 1 integrin immunoreactivity in point contacts. Immunoreactivity to p125FAK was not detected in PC12 cells, although astrocytes, which have both focal contacts and point contacts have p125FAK only at focal contacts. These observations, together with previous data (Turner et al., 1989; Tawil et al., 1993), suggest that point contacts are functional adhesion sites and are structurally distinct from focal contacts found in non-neuronal cells.

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“…antibodies alone are ineffective, whereas antibodies to al partially inhibit neurite extension (Turner et al, 1989;Tomaselli et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

Up-regulation of the integrin alpha 1/beta 1 in human neuroblastoma cells differentiated by retinoic acid: correlation with increased neurite outgrowth response to laminin.

et al. 1991

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Retinoic acid (RA) is known to induce differentiation of neuroblastoma cells in vitro. Here we show that treatment of two human neuroblastoma cell lines, SY5Y and IMR32, with RA resulted in a fivefold increase of the integrin alpha 1/beta 1 expression. The effect was selective because expression of the alpha 3/beta 1 integrin, also present in these cells, was not increased. The up-regulation of the alpha 1/beta 1 differentiated SY5Y cells correlated with increased neurite response to laminin. In fact, RA-treated SY5Y cells elongated neurites on laminin-coated substratum more efficiently compared with untreated cells or cells treated with nerve growth factor, insulin, or phorbol 12-myristate 13-acetate. These three agents induced partial morphological differentiation but did not increase alpha 1 integrin expression. Neurite extension in RA-treated cells was more efficient on laminin than on fibronectin or collagen type I and was inhibited with beta 1 integrin antibodies on all three substrates. Affinity chromatography experiments showed that alpha 1/beta 1 is the major laminin receptor in both untreated and RA-treated SY5Y cells. These data show that RA, a naturally occurring morphogen implicated in embryonic development, can selectively regulate the expression of integrin complexes in neuronal cells and suggest an important role of the alpha 1/beta 1 laminin receptor in the morphological differentiation of nerve cells.

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Identification of a cell-surface protein involved in PC12 cell- substratum adhesion and neurite outgrowth on laminin and collagen

Cited by 135 publications

References 58 publications

.alpha.1.beta.1 Integrin heterodimer functions as a dual laminin/collagen receptor in neural cells

.alpha.1.beta.1 Integrin heterodimer functions as a dual laminin/collagen receptor in neural cells

Characterization of neural cell adhesion sites: point contacts are the sites of interaction between integrins and the cytoskeleton in PC12 cells

Up-regulation of the integrin alpha 1/beta 1 in human neuroblastoma cells differentiated by retinoic acid: correlation with increased neurite outgrowth response to laminin.

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