Integrin-Mediated Tyrosine Phosphorylation and Redistribution of Paxillin during Neuronal Adhesion (original) (raw)
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Journal of cell science, 1994
Integrin alpha 6 beta 1 is a laminin receptor involved in adhesion and neurite extension of retinal neurons on laminin. The present study was carried out to identify potential interactions between the alpha 6 beta 1 receptor and cellular proteins that may be involved in integrin signaling and function. For this purpose we have used a biochemical approach based on the solubilization of retinal neurons cultured on laminin with nonionic detergents, followed by centrifugation on sucrose velocity gradients. Analysis of the distribution of the alpha 6 and beta 1 integrin subunits in the gradients showed that they migrate as a large complex after extraction of cells with octylglucoside, but not after Triton X-100 extraction. Cytoskeletal proteins known to localize in adhesion plaques did not comigrate with alpha 6 beta 1 in octylglucoside gradients, while a set of polypeptides whose tyrosine phosphorylation was enhanced by culture on laminin colocalized with alpha 6 beta 1 on the gradients...
The Journal of Cell Biology, 1987
Neuronal responses to extracellular matrix (ECM) constituents are likely to play an important role in nervous system development and regeneration. We have studied the interactions of a neuron-like rat pheochromocytoma cell line, PC12, with ECM protein-coated substrates. Using a quantitative cell attachment assay, PC12 cells were shown to adhere readily to laminin (LN) or collagen IV (Col IV) but poorly to fibronectin (FN). The specificity of attachment to these ECM proteins was demonstrated using ligandspecific antibodies and synthetic peptides. To identify PC12 cell surface proteins that mediate interactions with LN, Col IV, and FN, two different antisera to putative ECM receptors purified from mammalian cells were tested for their effects on PC12 cell adhesion and neuritic process outgrowth. Antibodies to a 140-kD FN receptor heterodimer purified from Chinese hamster ovarian cells (anti-FNR; Brown, P. J., and R. L. , J. Cell Biol., 103:1595-1603 inhibited attachment to LN and FN but not to Col IV.
Investigative Ophthalmology & Visual Science, 2006
Purpose-Retinal diseases are often accompanied by changes in the structure of the multilayered extracellular matrix underlying the retina, Bruch's membrane (BrM). These structural revisions potentially lead to alterations in retinal pigment epithelium (RPE) adhesion, likely via modification of interactions with extracellular matrix (ECM) proteins including laminins in BrM. The purpose of this study was to identify specific laminins in BrM and their receptors in RPE cells.
Ligand-induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth
Nature, 1997
Receptors of the integrin family are expressed by every cell type and are the primary means by which cells interact with the extracellular matrix. The control of integrin expression affects a wide range of developmental and cellular processes, including the regulation of gene expression, cell adhesion, cell morphogenesis and cell migration. Here we show that the concentration of substratum-bound ligand (laminin) post-translationally regulates the amount of receptor (alpha6beta1, integrin) expressed on the surface of sensory neurons. When ligand availability is low, surface amounts of receptor increase, whereas integrin RNA and total integrin protein decrease. Ligand concentration determines surface levels of integrin by altering the rate at which receptor is removed from the cell surface. Furthermore, increased expression of integrin at the cell surface is associated with increased neuronal cell adhesion and neurite outgrowth. These results indicate that integrin regulation maintain...
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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 ...
Inhibition of integrin-mediated adhesion and signaling disrupts retinal development
Developmental Biology, 2004
Integrins are the major family of cell adhesion receptors that mediate cell adhesion to the extracellular matrix (ECM). Integrin-mediated adhesion and signaling play essential roles in neural development. In this study, we have used echistatin, an RGD-containing short monomeric disintegrin, to investigate the role of integrin-mediated adhesion and signaling during retinal development in Xenopus. Application of echistatin to Xenopus retinal-derived XR1 glial cells inhibited the three stages of integrin-mediated adhesion: cell attachment, cell spreading, and formation of focal adhesions and stress fibers. XR1 cell attachment and spreading increased tyrosine phosphorylation of paxillin, a focal adhesion associated protein, while echistatin significantly decreased phosphorylation levels of paxillin. Application of echistatin or h 1 integrin function blocking antibody to the embryonic Xenopus retina disrupted retinal lamination and produced rosette structures with ectopic photoreceptors in the outer retina. These results indicate that integrin-mediated cell-ECM interactions play a critical role in cell adhesion, migration, and morphogenesis during vertebrate retinal development.
Differential outgrowth of retinal neurites on purified extracellular matrix molecules
Journal of Neuroscience Research, 1988
Organotgpic cultures of the embryonic retina were INTRODUCTION Development of the visual system involves a numsystem* Microexplants from the of a projection of axons from the retinal ganglion cells to the visual centers in the brain. The cellular and molecular during There is evidence that retinal ganglion cells can be No fiber Outgrowth occurred On substrata Of trophically stimulated by soluble factors extracted from of the brain lNurcombe and Bennett, promoted On laminin in a dose-dependent ler, 1982; manner. Fibronectin elicited a neurite outgrowth cor- Turner et al, 19831. In addition, the availability of a one-third the length Of the Out-suitable substratum has been shown to play an important growth On laminin' A 319000-da1ton fibronectin role in neuronal survival, maturation, and neurite extenmoted by the intact fibronectin molecule. Other isoof the formation of the visual system it lated domains Of fibronectin, including the 1059000to identify extracellular matrix molecules responsible for dalton "cell-binding" domain, did not allow neurite the elongation and guidance of growing fibers. outgrowth. Furthermore, preincubation of fibronec-Earlier investigations have presented evidence that tin substratum with antibodies to the heparin-binding at least two extracellular matrix proteins, fibronectin used to study the influence of extracellular matrix Of the chick retina (embryonic day 6, were grown in medium matrix molecules adsorbed to plastic at various conguidance of the optic axOnS to their target centrations. The maximum neurite length obtained on these processes are incompletely understood. each type of substratum was measured on day 4 of vitronectin or a hyaluronate-binding chondroitin sul-molecules On neurite during ber of cellular interactions leading to the containing appropriate trophic On purified mechanisms underlying neurite extension and selective the visual fate proteoglycan* In contrast, neurite was 1981; Schwam and Agranoff, 1981; Hyndman and Adto fragment elicited neurite elongation the heParin-binding domain sion [Letourneau, 1982; Thomson and Pelto, 1982; Edto that progar, 19851. In order to understand the molecular aspects necessary fibronectin fragment entirely Prevented ("'tgrowth. (FN) and laminin (LN), are potent promoters of neurjte Fiber Outgrowth was evoked On substrata Of elongation in vitro [Akers et al, 1981; Baron-Van Ever-Platelet factor 4, a protein binding heparan cooren et al, 1982; Carbonetto et al, 1983; Manthorpe et Adding increasing concentrations of heparin Progresal, 1983; Rogers et d, 1983, 1985; Faivre-Bauman et al, sively inhibited the neurite extension on laminin, 1984; Smalheiser et al, 1 9 a ; Adler et al, 1985; Calof whereas similar addition of soluble chondroitin sulfate and Reichafi, 1985; Davis et al, 1985a,b; Lander et al, Proteogbcan had no effect' The growing retinal neurites show strong preference for respond somewhat differently to these proteins. indicate that 19851. However, neurons of different origin Seem to Fibronectin has been reported to promote neurite laminin versus fibronectin. Moreover, the outgrowthto be localized to their heparin-binding regions. It is but not of neurons from the central system [cUlptern, elongating retinal neurites can actively discrimiined the capacity of retinal neurites to extend on different nate between different extracellular molecules by a matrix molecules when provided with optimal trophic mechanism that may involve participation of cell surface heparan sulfate proteoglycans. Promoting Of both adhesion Proteins outgrowth of neurons from the peripheral nervous system suggested that during Of the ' YSet d, 19861. In the present investigation we have exam-
alpha.1.beta.1 Integrin heterodimer functions as a dual laminin/collagen receptor in neural cells
Biochemistry, 1990
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 (Turner et al., 1987; Tomaselli et al., 1987, 1988). These functional studies suggested that PC12 cells possess a dual laminin/collagen receptor (Turner et al., 1987). Subsequently, monoclonal antibody (mab) 1 3A3 generated against PC12 cells was found to inhibit adhesion to and neurite outgrowth on laminin and collagen (Turner et al., 1989). mab 3A3 immunoprecipitates two proteins of ~ 110 and ~ 185 kDa. First, functional (Turner et al., 1989) and, later, immunochemical data suggested these proteins were members of the integrin family (Hynes,
Regulation of neurite outgrowth by integrin activation
The Journal of …, 2000
During late-embryonic development, retinal neurons lose the ability to attach and extend neurites on the extracellular matrix molecule laminin-1 (LN-1), despite the fact that they retain expression of integrin receptors for LN-1. Here we show that the developmental loss of responsiveness to LN-1 can be reversed by treatments that increase the activation state of integrins. Both extracellular application of Mn 2ϩ (at micromolar concentrations) and viral-mediated neuronal expression of a constitutively active form of the ras-related GTPase R-ras (R-ras 38V ) potently promoted late-embryonic retinal neurite outgrowth on LN-1 substrata. In both cases, outgrowth was mediated by integrin ␣61 and not ␣31, even though these neurons express ␣31 and use it for outgrowth on other laminin isoforms, as well as on LN-1 that has been proteolytically or conformationally activated . Mn 2ϩ -and to a much lesser extent R-ras 38V -also re-versed the developmental loss of retinal neuron responsiveness to type IV collagen, by promoting the function of integrin ␣11. Interestingly, the responses of other late-embryonic CNS neurons to LN-1 were also enhanced by treatments that activate integrin function, but those of peripheral nervous system neurons (dorsal root ganglion neurons) were either not enhanced (embryonic neurons) or only modestly improved (adult neurons). These results suggest that a developmental decline occurs in the activation state of neuronal integrins, particularly among CNS neurons. Such a decline may underlie some of the intrinsic loss of regenerative ability sustained by CNS neurons during development and may be a valid target for therapeutic intervention.
L1/HNK-1 Carbohydrate- and �1 Integrin-Dependent Neural Cell Adhesion to Laminin-1
J Neurochem, 2002
We have shown recently that mouse small cerebellar neurons adhere to a short amino acid sequence of the G2 domain of the laminin al chain via the cell surface-expressed HNK-i carbohydrate. Therefore, we were interested in identifying glycoproteins carrying the HNK-1 carbohydrate at the cell surface of these neurons. Adhesion of small cerebellar neurons to laminin is partially dependent on Ca 2~,Mn2-, and Mg2~,indicating the involvement of integrins, which were identified as~3i,a3, and a6. They could be shown to bind to laminin by a /31dependent adhesion mechanism. None of these subunits was found to carry the HNK-1 carbohydrate. HNK-1-immunoreactive glycoproteins were immunoprecipitated and shown to consist of predominantly one molecular species, which was identified as the neural cell recognition molecule Li. Li was demonstrated to bind in a concentration-dependent and saturating manner to laminin. The binding could be partially inhibited by Fab fragments of monoclonal antibodies against the HNK-1 carbohydrate and against the Ig-like domains of Li. Furthermore, antibodies to the Ig-like domains of Li and~31integrin inhibited partially cell adhesion to laminin. Determination of the association of Li,~31integrin, and the HNK-i carbohydrate on the cell surface of live cerebellar neurons by antibody-induced patching and copatching revealed HNK-i to be linked to Li, but less so to /31 integrin. However, only negligible association was found between Li and /31 integrin. Furthermore, it could be shown that adhesion to laminin is mediated by Li/HNK-i-and /3i integrin-dependent mechanisms that act at least partially independent of each other.