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At least 19 recordsLinked to original sources
PubMed1992

Chicken vinculin and meta-vinculin are derived from a single gene by alternative splicing of a 207-base pair exon unique to meta-vinculin.

meta-Vinculin and vinculin are closely related proteins that are cytoplasmic components of microfilament-associated cell junctions. This report describes the structural relationship between these two proteins and the genetic basis for tissue-specific expression of meta-vinculin. Analysis of genomic DNA coding for amino acids 676-1066 of vinculin revealed 9 exons spanning an 11.7-kilobase pair region of the genome. In the 4 kilobase pairs of intervening sequence that separates vinculin exons E896-E915 and E916-E984, there is an open reading frame that predicts a sequence homologous to the 68-amino acid peptide specific to porcine meta-vinculin (Gimona, M., Small, J. V., Moeremans, M., Van Damme, J., Puype, M., and Van-dekerckhove, J. (1988) EMBO J. 7, 2329-2334). Analysis of the corresponding cDNA established that chicken meta-vinculin contains a 69-amino acid insertion between residues 915 and 916 of vinculin and that there are no other amino acid sequence differences between chicken vinculin and meta-vinculin. Muscle-specific expression of meta-vinculin occurs by alternative splicing of a transcript produced from a single gene because: all 20 genomic isolates that contain the 3' vinculin exons, also contain the meta-vinculin-specific exon; Southern blots performed at high stringency with exon-specific probes indicate the presence of a single gene; and the 3'-untranslated sequences of vinculin and meta-vinculin cDNAs are identical.

Amino Acid Sequence↗
PubMed1992

An additional exon in the human vinculin gene specifically encodes meta-vinculin-specific difference peptide. Cross-species comparison reveals variable and conserved motifs in the meta-vinculin insert.

We have analyzed the structure, origin and expression of the high-molecular-mass muscle-specific variant of vinculin, called meta-vinculin. The meta-vinculin-specific inserts from the human and avian molecules have been isolated and sequenced and the sequences confirmed via cloning of the corresponding cDNA. Comparison of the human, avian and determined porcine sequences revealed cross-species identity in the C-terminal half of the insert. Human and porcine meta-vinculin were highly similar in the insert region, showing only five amino acid exchanges; avian meta-vinculin showed 22 exchanges in the same region compared to human meta-vinculin and exhibited, in addition, one extra amino acid, making 69 in all. Each insert was flanked by characteristic KWSSK motifs. Evidence for two vinculin mRNA species in human uterus smooth muscle was provided by reverse transcription combined with the polymerase chain reaction, as well as by ribonuclease-mapping analysis of cDNA/mRNA hybrids. One of the mRNA species contained an additional 204-nucleotide insert that precisely encoded the meta-vinculin-specific peptide. Sequence analysis of the appropriate portion of the human vinculin gene showed that the section coding for the meta-vinculin-specific insert is present as a discrete exon. Thus, meta-vinculin and vinculin mRNA are generated by alternative splicing.

Amino Acid Sequence↗
PubMed1983

Vinculin phosphorylation by the src kinase. Interaction of vinculin with phospholipid vesicles.

Vinculin phosphorylation by pp60src is stimulated by anionic phospholipids (Ito, S., Richert, N., and Pastan, I. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4628-4631). We have examined whether vinculin interacts with phospholipids, the specificity of the interactions, and a possible mechanism for the enhancement of vinculin phosphorylation by these phospholipids. 3H-labeled vinculin binds to phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. No binding to phosphatidylcholine or phosphatidylethanolamine was observed. The phospholipid binding specificity correlated with the ability of these phospholipids to enhance vinculin phosphorylation by the src kinase. Chlorpromazine (0.1 and 0.3 mM) inhibited both vinculin binding to phosphatidylinositol and the enhanced phosphorylation of vinculin by pp60src in the presence of phosphatidylinositol. Tryptic peptide maps of vinculin phosphorylated in the absence of phospholipid revealed three phosphorylated peptides. The same three peptides were phosphorylated in the presence of phospholipid. However, phosphorylation at one site was markedly increased. In the presence of phospholipid proteolysis of vinculin with both chymotrypsin and V8 protease was markedly enhanced and different peptide maps of vinculin were generated. Microheterogeneity of vinculin was observed with isoelectric focusing. All the isoforms (pI 5.45-5.8) were found to bind phospholipids and undergo phosphorylation by the src kinase. These results suggest that one way anionic phospholipids can enhance vinculin phosphorylation is by binding to vinculin and inducing a conformational change in the vinculin molecule.

Animals↗
PubMed1987

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Vinculin is part of the cadherin-catenin junctional complex: complex formation between alpha-catenin and vinculin.

In epithelial cells, alpha-, beta-, and gamma-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. alpha-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell-matrix and cell-cell contacts, alpha-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell-cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and alpha-catenin. We show that alpha-catenin colocalizes at cell-cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to alpha-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The Kd of the complex was determined to 2-4 x 10(-7) M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of alpha-catenin is involved in this interaction. Complex formation of vinculin and alpha-catenin was challenged in transfected cells. In PtK2 cells, intact alpha-catenin and alpha-catenin1-670, harboring the beta-catenin- binding site, were directed to cell-cell contacts. In contrast, alpha-catenin697-906 fragments were recruited to cell-cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo alpha-catenin, like vinculin, is tightly regulated in its ligand binding activity.

Animals↗
PubMed1983

Detection of vinculin-binding proteins with an 125I-vinculin gel overlay technique.

Vinculin is an adhesion plaque component localized on the cytoplasmic side of the cell membrane where stress fibers end. To detect vinculin-binding proteins, we have developed an 125I-vinculin gel overlay method. SDS PAGE was used to separate different protein preparations. After fixing the proteins in the gel with methanol-acetic acid, the SDS was removed with ethanol and the proteins renatured in buffer. The gel was then incubated with 125I-vinculin. After extensive washing to remove nonspecifically associated label, the gel was dried and autoradiographed. Chick embryo fibroblasts, their Rous sarcoma virus transformants, and HeLa cells were found to contain two proteins (Mr 220,000 and 130,000) that bound 125I-vinculin strongly and another (Mr 42,000) that bound it moderately. The 130,000-mol-wt protein was identified as vinculin itself, which suggests that it may self-associate. The 42,000-mol-wt protein was identified as actin with which vinculin is known to interact. The identity of the 220,000-mol-wt protein is not known. It is not cellular fibronectin, myosin, or filamin. When fibroblast proteins were separated into Triton X-100 soluble and insoluble fractions, most of the vinculin and the 220,000-mol-wt protein was found to be in the soluble fraction. Chicken gizzard also contained these vinculin-binding proteins along with three others of Mr 190,000, 170,000, and 100,000.

Animals↗
PubMed1994

Intramolecular interactions in vinculin control alpha-actinin binding to the vinculin head.

Using blot overlay techniques we have investigated the interaction of vinculin with alpha-actinin. We show that an alpha-actinin binding site is located in the 90 kDa vinculin head and confirm a vinculin binding site in the C-terminal rod of alpha-actinin, as recently reported by McGregor et al. [(1994) Biochem. J. 310, 225-233]. The isolated vinculin head binds much more strongly to alpha-actinin than intact vinculin. Using a proteolytic 81 kDa head fragment, we show that vinculin residues 1-107 are required for alpha-actinin binding. Antibodies directed against vinculin residues 808-850 inhibit the vinculin-alpha-actinin binding, suggesting that this sequence is directly involved in, or topographically related to, the alpha-actinin binding site.

Actinin↗
PubMed1998

Rescue of the mutant phenotype by reexpression of full-length vinculin in null F9 cells; effects on cell locomotion by domain deleted vinculin.

Vinculin plays a role in signaling between integrins and the actin cytoskeleton. We reported earlier that F9-derived cells lacking vinculin are less spread, less adhesive, and move two times faster than wild-type F9 cells. Expression of intact vinculin in null cells restored all wild-type characteristics. In contrast, expression of the head (90 kDa) fragment exaggerated mutant characteristics, especially locomotion, which was double that of vinculin null cells. Expression of the tail domain also had a marked effect on locomotion in the opposite direction, reducing it to very low levels. The expression of the head plus tail domains together (no covalent attachment) effected a partial rescue towards wild-type phenotype, thus indicating that reexpressed polypeptides may be in their correct location and are interacting normally. Therefore, we conclude that: (1) the head domain is part of the locomotory force of the cell, modulated by the tail, and driven by the integrin/matrix connection; (2) intact vinculin is required for normal regulation of cell behavior, suggesting that vinculin head-tail interactions control cell adhesion, spreading, lamellipodia formation and locomotion.

Cell Line↗
PubMed1982

Co-existence of vinculin and a vinculin-like protein of higher molecular weight in smooth muscle.

Recently, a protein component of adhesion plaques with a molecular weight of 130,000 (named vinculin) has been purified from smooth muscle and non-muscle cells. As detected by immunological methods, the only vinculin-related polypeptides in fibroblasts are proteins of Mr = 130,000. However, we show here that smooth muscle contains, in addition to vinculin, an apparently distinct protein with a Mr = 152,000 that shares both structural and immunological features with vinculin. Amino acid analysis, peptide mapping, and antibody cross-reaction studies were used to elucidate these similarities. Mr = 152,000 protein seems to be restricted to muscle (mainly or exclusively to smooth muscle). The possibility that vinculin is derived from proteolytic processing of the Mr = 152,000 protein or that the proteins are related by some other type of post-translational modification appears unlikely (although this cannot be completely ruled out) since both proteins are made in a rabbit reticulocyte cell-free translation system when mRNA derived from smooth muscle is used as the template. Both proteins are capable of used as the template. Both proteins are capable of lowering the viscosity of F-actin solutions, although the activity of the Mr = 152,000 protein is stimulated by Ca2+ while the activity of smooth muscle vinculin is not.

Amino Acids↗
PubMed1993

Organization of the human gene encoding the cytoskeletal protein vinculin and the sequence of the vinculin promoter.

The human vinculin gene contains 22 exons ranging in size from 71 base pairs (bp) to 303 bp (average 155 bp) with the exception of exon 22 which contains 144 bp of coding sequence and 1848 bp of 3'-untranslated sequence including two polyadenylation signals. There is a limited correlation between exon boundaries and functional domains within the vinculin molecule. The talin-binding domain in vinculin spans residues 1-258, and the first 6 exons encode residues 1-261. Similarly, the predicted boundaries of the central repeat domain (residues 259-589) are close to the boundaries of exons 7 and 12. Analysis of vinculin mRNAs in human uterus showed that alternative splicing of the gene is limited to exon 19, which encodes the 68 amino acids included in the muscle-specific isoform called metavinculin. We have determined 1.1 kilobases of sequence 5' of the transcription start site. The vinculin promoter lacks a TATA box, but does contain six Sp1 sites, and a CArG box at position -262 which forms the core of the serum response element found in immediate-early response genes. Expression of a vinculin promoter/CAT construct is serum-inducible in NIH3T3 cells demonstrating that the promoter does contain a functional serum response element.

3T3 Cells↗
PubMed1985

Electron microscopy of rotary shadowed vinculin and vinculin complexes.

Chicken gizzard smooth muscle vinculin, purified according to the method of Feramisco & Burridge (1980), was examined by rotary shadowing and electron microscopy. Individual vinculin molecules have two domains: a globular head with a diameter of 8.0 nm, and a tail 20 nm long. In high salt, vinculin self-associates into multimers containing two to six individual molecules. These molecules associate head to head and tail to tail, but the tail to tail association appears to be favored. Electron microscopy of the approximately 100,000 Mr major fragment of vinculin was performed. The tail region appeared to be cleaved off, making the head region less compact.

Animals↗
PubMed1988

Immunolocalization of meta-vinculin in human smooth and cardiac muscles.

Meta-vinculin, a vinculin-related protein, has been isolated from human uterus smooth muscle. Specific antibodies to meta-vinculin, which distinguish between meta-vinculin and vinculin, were prepared by absorption of anti-meta-vinculin serum on vinculin coupled to nitrocellulose. Meta-vinculin specific antibody demonstrates only smooth and cardiac muscle specificity and is able to cross-react with a small 21-kD fragment of the meta-vinculin polypeptide chain. This antibody does not interact with protease resistant 95-kD core shared by vinculin and meta-vinculin. Meta-vinculin specific antibody was used for the localization of meta-vinculin in smooth and cardiac muscles by the indirect immunofluorescence method. At the light microscopy resolution level it was found that meta-vinculin and vinculin are localized in the same cellular adhesive structures. Meta-vinculin is present in membrane-associated microfilament-bound plaques of smooth muscle, in intercalated discs and costameres of cardiac muscle. In primary culture of smooth muscle cells from human aorta, meta-vinculin and vinculin were found to be present in focal contacts of the cells. During the cultivation of smooth muscle cells, the quantity of meta-vinculin decreased progressively and finally meta-vinculin completely disappeared from the focal contacts. The data show that in smooth and cardiac muscles meta-vinculin could be a structural component of microfilament-membrane attachment sites, defined earlier by the localization of vinculin.

Antibody Specificity↗
PubMed1988

Diversity of vinculin/meta-vinculin in human tissues and cultivated cells. Expression of muscle specific variants of vinculin in human aorta smooth muscle cells.

Microheterogeneity of different vinculin and meta-vinculin isoforms in adult human tissues and cultured cells was studied by two-dimensional gel electrophoresis and immunoblotting technique. Four isoforms of vinculin (alpha, alpha', beta, and gamma) and two isoforms of meta-vinculin (alpha and beta) were resolved. alpha-, alpha'-, and beta-isoforms of vinculin were found in all cell types and tissue samples analyzed in the present study. gamma-Isoform of vinculin and both alpha- and beta-isoforms of meta-vinculin were found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, foreskin fibroblasts, and macrophages. In the primary culture of human aorta smooth muscle cells, the fractional content of gamma-isoform of vinculin and meta-vinculin was dramatically reduced, and, by the onset of intensive cell division, the proteins could hardly be detected. Subcultured human aorta smooth muscle cells did not contain gamma-vinculin and meta-vinculin. We analyzed the microheterogeneity of vinculin and meta-vinculin in three smooth muscle layers of human aorta wall--media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. It was shown that in media the fractional content of gamma-isoform of vinculin was 45% and meta-vinculin, 42%; in muscular-elastic intima the fractional content of gamma-vinculin was 42% and meta-vinculin, 36%. However, in subendothelial intima, the share of these proteins was significantly lower than in adjacent muscular-elastic intima and media. Isoactin pattern that is characteristic of smooth muscle was identical in all aortic layers, thus proving the smooth muscle origin of subendothelial intima cells. These findings demonstrate that human aortic smooth muscle cells in vivo and in vitro undergo coordinated differential expression of smooth muscle specific variants of vinculin, i.e. gamma-vinculin and meta-vinculin.

Adult↗
PubMed1987

Properties of smooth muscle meta-vinculin.

Quantitative studies show that meta-vinculin is ninefold more soluble in 0.6 M salt than in the 0.01 M salt buffers used to extract vinculin. Based on this finding, we have developed a protocol for the purification of meta-vinculin in 43% yield and 98% purity from a high salt extract of gizzard smooth muscle. In contrast to our earlier extraction studies, which were done on unfixed cryostat sections (30), the present studies done on tissue homogenates show that nonionic detergents are not required for solubilization of meta-vinculin. Furthermore, neither purified nor partially purified meta-vinculin binds to Triton X-114 micelles. Purified meta-vinculin is a monomeric, asymmetric molecule with a Stokes radius of 50.9 A, a sedimentation coefficient of 6.35S, and a frictional ratio of 1.46. The calculated molecular weight of meta-vinculin is 145,000. Meta-vinculin has two isoforms of pI 5.9 and 6.2, and is phosphorylated in vivo to eightfold greater specific activity than vinculin. On immunoblots of smooth muscle proteins, [125I]meta-vinculin binds specifically to talin and also to unidentified polypeptides of 180, 150, 95, 70, 68, and 45 kD. On two-dimensional peptide maps, iodinated vinculin and meta-vinculin have at least 95% of their major chymotryptic peptides in common, but each protein also has at least one highly labeled peptide that appears to be unique. Comparative peptide maps of high salt soluble meta-vinculin and the low salt soluble 152-kD protein (described by Feramisco, J.R., J.E. Smart, K. Burridge, D. Helfman, and G.P. Thomas, 1982, J. Biol. Chem., 257:11024-11031) demonstrate extensive similarities among the vinculin-like proteins but suggest a lack of complete identity. In vivo pulse-chase experiments show that meta-vinculin and vinculin do not have a precursor-product relationship. The biochemical and structural differences found between vinculin and meta-vinculin suggest that there is a unique function for meta-vinculin in smooth muscle.

Animals↗
PubMed1986

Identification of the cellular mechanisms responsible for platelet-derived growth factor induced alterations in cytoplasmic vinculin distribution.

Exposure of quiescent density arrested BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF; 6-12 ng/ml) results in a rapid, reversible, time- and dose-dependent removal of vinculin from adhesion plaques (Herman and Pledger, 1985). Potential cellular mechanisms involved in PDGF-induced removal of vinculin from adhesion plaques were examined. Removal of vinculin from adhesion plaques following exposure of cells to PDGF was temperature dependent, occurred in many fibroblast cell lines, and could be mimicked by 12-tetradecanoyl phorbol-13-acetate (TPA; 5-125 nM) or melittin (0.35 microM). Unlike the effect of PDGF, TPA- or melittin-induced vinculin disruption was not reversible. The removal of vinculin from adhesion plaques was inhibited by trifluoroperazine (TFP; 2.5 microM). 8-(N,N-diethylamino) octyl-3,4,5-trimethoxy benzoate (TMB-8; 1.0 microM), mepacrine (220 microM), n-alpha-p-tosyl-L-lysine chloromethylketone (TLCK; 100 microM), phenylmethoxysulphonylfluoride (PMSF; 500 microM), and epsilon-aminocaproic acid (epsilon-ACA; 100 microM); however, amiloride (100 microM), A23187 (20 microM), and chloroquine (1 mM) were unable to inhibit this effect. Melittin disruption of vinculin was inhibited by (in order of decreasing effectiveness) mepacrine greater than TMB-8 greater than TFP greater than leupeptin greater than PMSF, whereas A23187 and amiloride had no effect. The return of vinculin to adhesion plaques following PDGF treatment required de novo mRNA transcription and protein synthesis and was associated with PDGF-stimulated synthesis of vinculin. The observation that both PDGF- and melittin-induced removal of vinculin from adhesion plaques is inhibited by mepacrine suggests that phospholipase activation may be an early and important step in PDGF-induced disruption of vinculin from adhesion plaques. In addition, TFP, TMB-8 and protease inhibitor inhibition of both the PDGF and melittin effects on vinculin distribution, coupled with the finding that TPA can mimic the PDGF or melittin response, suggests that Ca2+, calmodulin, protein kinase C, and/or proteolysis may play an important role(s) in the removal of vinculin from adhesion plaques following PDGF addition. The lack of effect of A23187 addition on vinculin distribution suggests that alterations in cellular Ca2+ is necessary but not sufficient for vinculin removal from adhesion plaques.

Amiloride↗
PubMed1998

The interaction of the cell-contact proteins VASP and vinculin is regulated by phosphatidylinositol-4,5-bisphosphate.

BACKGROUND: Focal adhesion sites are cell-matrix contacts that are regulated by phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent pathways. Vinculin is a major structural component of these sites and is thought to be engaged in multiple ligand interactions at the cytoplasmic face of these contacts. Cytoplasmic vinculin is considered to be inactive due to its closed conformation involving intramolecular head-tail interactions. Recently, the vasodilator-stimulated phosphoprotein (VASP), a substrate of cyclic AMP-dependent or cyclic GMP-dependent kinases and a component of focal adhesion sites, was shown to bind to vinculin. RESULTS: VASP-vinculin complexes could be immunoprecipitated from cell lysates and, using immunofluorescence, both proteins were found to colocalize in nascent focal adhesions. Consistent with the view that vinculin must be activated at these sites, we found that PIP2, levels of which are elevated during the early stages of adhesion, bound to two discrete regions in the vinculin tail, disrupting the intramolecular head-tail interaction and inducing vinculin oligomerization. Vinculin-VASP complex formation was greatly enhanced by PIP2 and both the EVH1 and EVH2 domains of VASP participated in vinculin binding. CONCLUSIONS: Focal contact assembly involves interaction between VASP and vinculin, which is enhanced by PIP2-induced vinculin activation and oligomerization. Given that vinculin and VASP both bind to F-actin, vinculin-VASP complexes might bundle the distal ends of actin filaments in focal contacts. We propose that PIP2-dependent signalling modulates microfilament organization at cellular adhesion sites by regulating vinculin-VASP complexes.

Actins↗