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Biomedical subjects

D R Critchley

Publications and source records attributed to D R Critchley.

At least 37 records · Page 2Linked to original sources
PubMed1997

Further analysis of the role of spectrin repeat motifs in alpha-actinin dimer formation.

Protein constructs consisting of repeats 1-4, repeats 1-3 and repeats 2-4 of the rod domain of chicken alpha-actinin were expressed as fusion proteins in Escherichia coli. Based on the evidence of circular dichroism spectra and cooperative thermal unfolding profiles both truncated rod fragments were judged to have assumed the native structural fold. The thermal stabilities were in both cases significantly lower than that of the intact rod (repeats 1-4). Analyses by sedimentation equilibrium and velocity provided further evidence to show that fragment 1-4 is entirely dimeric in the concentration range of these experiments, resembling therefore the rod domain isolated by proteolytic digestion of native alpha-actinin. Fragment 2-4, and probably also 1-3, show concentration-dependent association, with dissociation constants, estimated by sedimentation equilibrium, in the 1-10 microM range. Thus, in confirmation of earlier work, all four repeats are required to generate a maximally stable anti-parallel dimer (Kd approximately 10 pM), suggesting the presence of binding sites in all of them to allow for aligned pairing.

Actinin↗
PubMed1997

Requirement for Rho in integrin signalling.

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.

3T3 Cells↗
PubMed1996

Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity.

Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.

3T3 Cells↗
PubMed1996

Binding of the cytoplasmic domain of intercellular adhesion molecule-2 (ICAM-2) to alpha-actinin.

Intercellular adhesion molecule-2 (ICAM-2) functions as a ligand for lymphocyte function-associated antigen-1 (LFA-1) and is involved in leukocyte adhesion. We studied intracellular associations of ICAM-2 using a peptide encompassing the cytoplasmic amino acids 231-254 as an affinity matrix. Among the proteins from placental lysates that bound to the peptide was alpha-actinin as demonstrated by immunoblotting. Purified, 125I-labeled alpha-actinin also bound to the peptide. Confocal microscopic analysis of Eahy926 cells demonstrated a colocalization of ICAM-2 and alpha-actinin. Of overlapping octapeptides covering the entire ICAM-2 cytoplasmic amino acids, ICAM-2241-248 bound alpha-actinin most avidly and effectively competed with the longer cytoplasmic peptide for binding. The site of interaction in alpha-actinin was studied using bacterially expressed alpha-actinin fusion proteins. Several constructs covering nonoverlapping regions of alpha-actinin bound to the ICAM-2 cytoplasmic peptide suggesting that multiple regions in alpha-actinin can mediate the interaction. These results, together with previously demonstrated interactions between alpha-actinin and the adhesion proteins ICAM-1, L-selectin, beta1- and beta2-integrins emphasize the role of alpha-actinin as a linker between cell surface adhesion molecules and the actin-containing cytoskeleton.

Actinin↗
PubMed1996

The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin.

In mammalian cells vasodilator-stimulated phosphoprotein (VASP) is localized to focal adhesions and areas of dynamic membrane activity where it is thought to have a role in actinfilament assembly. The proteins responsible for recruiting VASP to these sites within the cell are not known. The bacterial protein ActA binds VASP via a proline-rich motif that is very similar to a sequence in the proline-rich region of the focal-adhesion protein vinculin. We have examined the ability of VASP, synthesized using an in vitro transcription/translation system, to bind to a series of vinculin peptides expressed as glutathione S-transferase fusion proteins, and have shown that it binds specifically to the proline-rich region in vinculin. Using immobilized peptides corresponding to the two proline-rich motifs within this domain, the VASP-binding site was localized to proline-rich motif-l (residues 839-850). Binding to this motif was not affected by the phosphorylation state of VASP. The C-terminal region of VASP, which is known to be important in targeting VASP to focal adhesions, was shown to be required for binding. These results identify vinculin as a VASP-binding protein likely to be important in recruiting VASP to focal adhesions and the cell membrane.

Actins↗
PubMed1996

Acidic phospholipids inhibit the intramolecular association between the N- and C-terminal regions of vinculin, exposing actin-binding and protein kinase C phosphorylation sites.

Chick vinculin polypeptides expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins have been used to identify the sites involved in the intramolecular association between the 90 kDa N-terminal head and the 30 kDa C-terminal tail region of the vinculin molecule. Fusion proteins spanning vinculin residues 1-258 and 1-398, immobilized on glutathione-agarose beads, were shown to bind a C-terminal vinculin polypeptide spanning residues 881-1066 (liberated from GST by thrombin cleavage). However, the C-terminal polypeptide did not bind to a fusion protein spanning residues 399-881 or to itself. Binding was dependent on residues 167-207 within the N-terminal polypeptide, a sequence also essential for talin binding. Conversely, the 90 kDa head polypeptide was shown to bind to residues 1029-1036 in the tail region of vinculin. The association of the head and tail was inhibited by acidic, but not neutral, phospholipids. Pre-incubation of vinculin with acidic phospholipids exposed the binding site for F-actin and a phosphorylation site for protein kinase C. The phosphorylation site was located in the tail region of the vinculin molecule. These results raise the possibility that acidic phospholipids play a role in regulating the activity of vinculin and therefore the assembly of both cell-cell and cell-matrix adherens-type junctions.

Actins↗
PubMed1996

A novel dystrophin/utrophin-associated protein is an enzymatically inactive member of the phosphoglucomutase superfamily.

A 60-kDa protein localised in adherens-type cellular junctions, and previously called aciculin, has been found to interact with the cytoskeletal proteins dystrophin and utrophin [Belkin, A. M. & Burridge, K. (1995) J. Biol. Chem. 270, 6328-6337]. In this study, we report the complete sequence of this protein, and show that it is a novel member of the phosphoglucomutase (PGM) family of proteins. The PGM-related protein (PGM-RP), which contains 506 amino acids (55.6 kDa), is smaller than PGM1 (566 amino acids, 61 kDa). The active site consensus sequences of prokaryotic and eukaryotic mutases are not conserved in PGM-RP, a finding consistent with the lack of enzymatic activity of PGM-RP in vitro, and the absence of a phosphorylated intermediate in vivo. The organisation of the PGM-RP gene is essentially identical to that of PGM1. We propose that the PGM-RP gene, which we have mapped to human chromosome 9qcen-q13, evolved from the PGM1 gene, and encodes a protein with a structural rather than an enzymatic role. PGM-RP is expressed predominantly in muscle with the highest levels in smooth muscle. The significance of the interaction between dystrophin/utrophin and an increasing number of cytoplasmic proteins including PGM-RP remains to be explored.

Amino Acid Sequence↗
PubMed1996

Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.

We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

Actins↗
PubMed1995

Association of structural repeats in the alpha-actinin rod domain. Alignment of inter-subunit interactions.

Fragments of the rod domain of chicken alpha-actinin, which comprises four spectrin-like repeat sequences, have been prepared by expression in Escherichia coli. Electron microscopy reveals that all products containing three or four complete repeats are rod-like. Self-association of fragments was detected by chemical cross-linking and analytical equilibrium sedimentation. The intact rod domain forms a stable dimmer, which does not dissociate measurably in the accessible concentration range. Elimination of either terminal repeat (repeat 1 or repeat 4) greatly diminishes the extent of dimerisation. The fragment comprising repeats 1-3 dimerises appreciably, with an association constant estimated from the sedimentation equilibrium distribution of approximately 5 x 10(5) M-1. The fragment made up of repeats 2-4 dimerises to a small extent, but also forms aggregates at high concentrations. The results are most easily reconciled with an aligned structure for the rod domain in solution, in which repeat 1 associates with repeat 4 of the partnering chain, and repeat 2 with repeat 3, rather than with a staggered structure, in which one of the terminal repeats does not participate in dimerisation. Possible explanations for the apparent difference observed between the alpha-actinin rod structure in solution and in two-dimensional crystalline arrays are examined.

Actinin↗
PubMed1995

Localisation of the human gene encoding the cytoskeletal protein talin to chromosome 9p.

The cytoskeletal protein talin is localised on the cytoplasmic face of the integrin family of adhesion receptors in cellular junctions with the extracellular matrix. Using polymerase chain reaction amplification and DNA from a panel of human-rodent somatic cell hybrids, we have assigned the talin gene to chromosome 9p. Deletions in 9p have been implicated in a variety of cancers, including malignant melanoma, and the concept that talin might be a candidate tumour suppressor gene is discussed.

Animals↗
PubMed1995

Utrophin actin binding domain: analysis of actin binding and cellular targeting.

Utrophin, or dystrophin-related protein, is an autosomal homologue of dystrophin. The protein is apparently ubiquitously expressed and in muscle tissues the expression is developmentally regulated. Since utrophin has a similar domain structure to dystrophin it has been suggested that it could substitute for dystrophin in dystrophic muscle. Like dystrophin, utrophin has been shown to be associated with a membrane-bound glycoprotein complex. Here we demonstrate that expressed regions of the predicted actin binding domain in the NH2 terminus of utrophin are able to bind to F-actin in vitro, but do not interact with G-actin. The utrophin actin binding domain was also able to associate with actin-containing structures, stress fibres and focal contacts, when microinjected into chick embryo fibroblasts. The expressed NH2-terminal 261 amino acid domain of utrophin has an affinity for skeletal F-action (Kd 19 +/- 2.8 microM), midway between that of the corresponding domains of alpha-actinin (Kd 4 microM) and dystrophin (Kd 44 microM). Moreover, this utrophin domain binds to non-muscle actin with a approximately 4-fold higher affinity than to skeletal muscle actin. These data (together with those of Matsumura et al. (1992) Nature, 360, 588-591) demonstrate for the first time that utrophin is capable of performing a functionally equivalent role to that of dystrophin. The NH2 terminus of utrophin binds to actin and the COOH terminus binds to the membrane associated glycoprotein complex, thus in non-muscle and developing muscle utrophin performs the same predicted 'spacer' or 'shock absorber' role as dystrophin in mature muscle tissues. These data suggest that utrophin could replace dystrophin functionally in dystrophic muscle.

Actins↗
PubMed1994

The heat-labile enterotoxin of Escherichia coli binds to polylactosaminoglycan-containing receptors in CaCo-2 human intestinal epithelial cells.

The E. coli type I heat-labile enterotoxin (LT-I) shares considerable functional, structural, and immunological homology with cholera toxin (CT). Although the ganglioside GM1 is the sole receptor for CT, LT-I also appears to utilize additional, unique receptors on intestinal cells not recognized by CT. We characterized this second class of LT-I receptors using the human intestinal epithelial cell line, CaCo-2. CaCo-2 cells bound 8-fold more LT-I than CT, and some of these additional LT-I receptors appeared to be functional, as CT-B only partially inhibited LT-I activity at concentrations that completely inhibited CT activity. Membranes from unlabeled or [3H]galactose-labeled cells were incubated with toxin B subunits and extracted with Triton X-100, and the solubilized toxin B-receptor complexes were immunoabsorbed with anti-B bound to protein A-Sepharose. When organic extracts of the complexes were separated by thin-layer chromatography and overlayed with [125I]toxin, both toxins were found to bind only GM1. Separation of the complexes from [3H]galactose-labeled membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a series of galactoproteins specifically recognized by LT-I but not by CT. Similar proteins were detected on Western blots probed with [125I]toxin. LT-I activity on intact cells and binding to membranes and the above galactoproteins were enhanced by neuraminidase treatment even in the presence of CT-B. beta-1,4-Galactosidase and endo-beta-1,4-galactosidase, but not beta-1,3-galactosidase, significantly reduced LT-I binding. LT-I binding to fetuin and transferrin exhibited a similar glycosidase sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Sugars↗
PubMed1994

Analysis of the phasing of four spectrin-like repeats in alpha-actinin.

Selected fragments of the central rod of chicken gizzard alpha-actinin were expressed as fusion proteins in Escherichia coli, with the aim of determining the positions in the sequence of the four successive spectrin-like repeats that make up this domain. The criteria for an independently folding unit were resistance to proteolysis and the high alpha helicity characteristic of the native protein. Sequences containing repeats 1-4, 2-4, 3-4 and 4 all generated stable fragments on digestion with trypsin and/or thermolysin and N-terminal sequencing gave the most probable starting position of each repeat. The sequences of all four inferred repeats and the sequences of the entire rod, were separately expressed and were shown to assume a stable, protease-resistant fold in solution. The repeat boundaries established in this way differed from those originally deduced from sequence alignments; the N-terminal boundaries of the repeats were 14-24 residues nearer the C-terminus than predicted. The ability to express individual repeats should facilitate identification of the binding sites for the cytoplasmic domains of beta 1 integrins and intercellular cell adhesion molecule-1 which have been localised to the rod domain of alpha-actinin.

Actinin↗
PubMed1994

Identification of the vinculin-binding site in the cytoskeletal protein alpha-actinin.

Using low-speed sedimentation equilibrium we have established that vinculin binds to alpha-actinin with a Kd of 1.3 x 10(-5) M. Electron microscopy of negatively stained preparations of vinculin revealed spherical particles (diameter 11.2 nm; S.D. 1.7 nm, n = 21), whereas alpha-actinin appeared as a rod-shaped particle (length 33 nm; S.D. 3.3 nm, n = 23). Mixtures of the two proteins contained both 'lollipop'- and 'dumbell'-shaped particles which we interpret as either one or two spherical vinculin molecules associated with the ends of the alpha-actinin rod. We have further defined the vinculin-binding site in alpha-actinin using 125I-vinculin and a gel-blot assay in which proteolytic fragments of alpha-actinin and fragments of alpha-actinin expressed in Escherichia coli were resolved by SDS/PAGE and blotted to nitrocellulose. 125I-vinculin bound to polypeptides derived from the spectrin-like repeat region of alpha-actinin, but did not bind to the actin-binding domain. Binding was inhibited by a 100-fold molar excess of unlabelled vinculin. Using a series of glutathione S-transferase fusion proteins we have mapped the vinculin-binding site to a region toward the C-terminal end of the molecule (alpha-actinin residues 713-749). 125I-vinculin also bound to fusion proteins containing this sequence which had been immobilized on glutathione-agarose beads. The vinculin-binding site is localized in a highly conserved region of the molecule close to the first of two EF-hand calcium-binding motifs.

Actinin↗
PubMed1994

The kinetics of the interaction between the actin-binding domain of alpha-actinin and F-actin.

Measurement of the binding equilibrium for the interaction of alpha-actinin with F-actin is complicated by secondary reactions involving cross-linking and/or bundling of the actin filaments. To quantitate the initial binding event, we studied the interaction of the bacterially expressed actin-binding domain (ABD) of chick smooth muscle alpha-actinin with F-actin. Stopped-flow measurements revealed a quench in protein fluorescence and an enhancement in light scattering when ABD binds to F-actin yielding second order rate constants for association of 2 x 10(5), 1.8 x 10(6) and 4 x 10(6) M-1.s-1 at 5 degrees C, 15 degrees C and 25 degrees C, respectively. At the latter two temperatures the dissociation rate constants were 1.5 and 9.6s-1, giving equilibrium constants of 0.83 and 2.4 microM, respectively. Optical changes on mixing intact alpha-actinin with F-actin were dominated by secondary bundling events.

Actinin↗