The Fibronectin Domains of the Neural Adhesion Molecule TAX-1 Are Necessary and Sufficient for Homophilic Binding (original) (raw)
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Molecular and Cellular Neuroscience, 2002
Cell adhesion molecules of the immunoglobulin superfamily promote cell aggregation and neurite outgrowth via homophilic and heterophilic interactions. The transient axonal glycoprotein TAG-1 induces cell aggregation through homophilic interaction of its fibronectin repeats. We investigated the domains responsible for the neurite outgrowth promoting activity of TAG-1 as well as its interactions with other cell adhesion molecules. Binding experiments with Fc-chimeric proteins revealed that TAG-1 interacts with L1, NrCAM, and F3/contactin. The membrane-associated as opposed to the soluble form of TAG-1 behaves differently in these assays. We demonstrate that both the immunoglobulin as well as the fibronectin domains promote neurite outgrowth when used as substrates. Furthermore we investigated the putative role of L1 and NrCAM as the neuronal TAG-1 receptors mediating neurite extension. DRG neurons from L1-deficient mice were found to extend neurites on TAG-1 substrates and blocking NrCAM function did not diminish the TAG-1-dependent neurite outgrowth. These results indicate that neither L1 nor NrCAM are required for TAG-1elicited neurite outgrowth.
Molecular Brain Research, 2003
Cell adhesion molecules (CAMs) of the immunoglobulin superfamily (IgSF) exhibit restricted spatial and temporal expression profiles requiring a tight regulatory program during development. The rodent glycoprotein TAG-1 and its orthologs TAX-1 in the human and axonin-1 in chick are cell adhesion molecules belonging to the contactin/F3 subgroup of the IgSF. TAG-1 is expressed in restricted subsets of central and peripheral neurons, not only during development but also in adulthood, and is implicated in neurite outgrowth, axon guidance and fasciculation, as well as neuronal migration. In an attempt to identify the regulatory elements that guide the neuronal expression of TAG-1, we have isolated genomic clones containing 4 kb of the TAX-1 upstream sequence and used them to drive the expression of the LacZ reporter gene in transgenic mice. We demonstrate that this sequence includes elements not only sufficient to restrict expression to the nervous system, but also to recapitulate to a great extent the endogenous pattern of the TAG-1 expression in the developing CNS. D
The Journal of Cell Biology
Cellular interactions with fibronectin-treated substrata have a complex molecular basis involving multiple domains. A carboxy-terminal cell and heparin binding region of fibronectin (FN) is particularly interesting because it is a strong promoter of neurite outgrowth (Rogers, S. L., J. B. McCarthy, S. L. Palm, L. T. Furcht, and P. C. Letourneau, 1985. J. Neurosci. 5:369-378) and cell attachment (McCarthy, J. B., S. T. Hagen, and L. T. Furcht. 1986. J. Cell Biol. 102:179-188.). To further understand the molecular mechanisms of neuronal interactions with this region of FN, we screened two peptides from the 33-kD heparin binding fragment of the FN A chain, FN-C/H II (KNNQKSEPLIGRKKT) and CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem. 262:6886-6892), for their ability to promote B104 neuroblastoma cell-substratum adhesion and neurite outgrowth. Both FN-C/H U and CS1 promoted B104 cell attachment in a concentration-dependent and saturable manner, with attachment to FN-C/H II exceeding attachment to CS1. In solution, both exogenous FN-C/H II 1. Abbreviations used in this paper: CNS, central nervous system; ECM, extracellular matrix; FN, fibroneetin; KLH, keyhole limpet hemocyanin; OA, ovalbumin; PNS, peripheral nervous system.
European Journal of Biochemistry, 1991
We previously described the production of monoclonal antibodies against a preparation of membrane glycoproteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809-8151. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135). Gpl35 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 11 5 kDa following N-glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments. Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins. Cell adhesion phenomena constitute an important factor in the developing nervous system [l]. One of the first steps during brain morphogenesis is the projection of axons to their targets through a diverse and changing environment. The final result is an intricate pattern of neuronal cells of different types forming complex nets of interactions characteristic for the mature brain. The way these internal cell contacts are generated is unknown. The organization has been shown to be guided by diffusible tropic factors, molecules expressed in the extracellular matrix, and membrane glycoproteins expressed on cells [2]. The knowledge of such brain-specific glycoproteins is still limited. In the group of brain glycoproteins participating in cell adhesion, three distinct gene families seem to be involved, including (a) the calcium-dependent adhesion molecules, the cadherins [3], (b) a group of cell surface receptors, the integrins, which bind to arginine-glycine-aspartic acid (RGD) or related sequences [4, 51 and finally (c) a group of cell adhesion molecules (CAMS) which have sequences related to
Recent advances in research on fibronectin and other cell attachment proteins
Journal of Cellular Biochemistry, 1985
Fibronectin and other cell attachment proteins provide molecular models for beginning to unravel the complex interactions of the cell surface with the extracellular matrix. This area has been reviewed in considerable detail previously [I-lo]. Our brief review will therefore be selective rather than comprehensive, and it will focus on some recent generalizations about this class of proteins, as well as on recent advances in the molecular analysis of the functions of these proteins and their receptors. We shall also present various popular or provocative hypotheses and speculations about future work in the field.
A human brain glycoprotein related to the mouse cell adhesion molecule L1
The Journal of biological chemistry, 1988
We have employed monoclonal antibody 5G3, an antibody used to label human tumor cells of neural origin (Mujoo, K., Spiro, R.C., and Reisfeld, R. A. (1986) J. Biol. Chem. 261, 10299-10305), to isolate and characterize a large glycoprotein from normal adult human brain. This protein was compared to mouse L1 (Rathjen, F. G., and Schachner, M. (1984) EMBO J. 3, 1-10), a neural cell surface glycoprotein implicated predominantly in neurite-neurite interactions. On the basis of the following results the 5G3 antigen is considered to be the human homologue of mouse L1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both proteins share similar molecular masses of their carbohydrate-depleted or undepleted components. In tryptic fingerprint analyses of the iodinated L1 and 5G3 components, 65% of the resolved peptides comigrated. Comparison of NH2-terminal amino acid sequences revealed a high degree of homology between human 5G3 and mouse L1, with 11 of 15 residues being identical...
Genomics, 1993
The transient axonal glycoprotein (TAG-l) is a cell adhesion molecule that promotes neurite outgrowth and belongs to the immunoglobulin superfamily. We have isolated cDNAs encoding TAX1, the human homologue of TAG-1. Human TAXl shows a high degree of homology to rat TAX1 and less to its chick counterpart, axonin-1, with 91 and 75% identity at the amino acid level, respectively. The numbers of immunoglobulin (IgC2) domains and fibronectin repeats present in TAG-1 are conserved among the three species. The highest degree of conservation occurs in the second IgC2 domain (98% with the rat and 82% with the chick). The human homologue also contains a putative N-terminal signal sequence and a C-terminal bydrophobic sequence, suggestive of linkage to the cell membrane via phosphatidylinositol. In addition, the two mammalian TAG-1 proteins share the RGD tripeptide, a motif known to mediate recognition of fibronectin by integrins. In situ hybridization to human metaphase chromosomes maps the TAX1 gene encoding human TAG-1 to a single location on chromosome lq32.
Journal of Cellular Biochemistry, 1985
We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band l), 135,000 (band 2 ) , and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band l), pH 5.6 (band 2 ) , and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S . Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.
Proceedings of the National Academy of Sciences, 2004
The extracellular regions of adhesion proteins of the Ig superfamily comprise multiple, tandemly arranged domains. We used directforce measurements to investigate how this modular architecture contributes to the adhesive interactions of the neural cell adhesion molecule (NCAM), a representative of this protein class. The extracellular region of NCAM comprises five immunoglobulin and two fibronectin domains. Previous investigations generated different models for the mechanism of homophilic adhesion that each use different domains. We use force measurements to demonstrate that NCAM binds in two spatially distinct configurations. Igdomain deletion mutants identified the domains responsible for each of the adhesive bonds. The measurements also confirmed the existence of a flexible hinge that alters the orientation of the adhesive complexes and the intermembrane distance. These results suggest that a combination of multiple bound states and internal molecular flexibility allows for seque...
European Journal of Biochemistry, 1992
Axonin-I is an axon-associated cell adhesion molecule (AxCAM) of the chicken, which promotes neurite outgrowth by interaction with the AxCAM Ll(G4) of the neuritic membrane. Here we report the cloning and sequence determination of a cDNA encoding axonin-I, Peptides generated by enzymatic cleavage showed similarity to the AxCAM F11. Degenerated polymerase chain reaction (PCR) primers were designed and an axonin-I fragment was amplified from mRNA of embryonic retina. Screening of a cDNA library from embryonic brain resulted in the isolation of a 4.0-kb cDNA insert with an open reading frame of 3108 nucleotides. The deduced polypeptide of 1036 amino acids includes a putative hydrophobic N-terminal signal sequence of 23 or 25 amino acids and a C-terminal hydrophobic sequence of 29 amino acids which is suggestive of sequences serving as signal for the attachment of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) anchor. The putative mature form of axonin-1 comprises six immunoglobulin-like repeats, followed by four fibronectin-type I11 repeats.