scholarly journals Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association

1992 ◽  
Vol 117 (6) ◽  
pp. 1321-1330 ◽  
Author(s):  
AA Reszka ◽  
Y Hayashi ◽  
AF Horwitz
Keyword(s):  
Amino Acids ◽  
Focal Adhesion ◽  
Point Mutations ◽  
Focal Adhesions ◽  
Alpha Helix ◽  
Cytoplasmic Domain ◽  
Amino Acid Sequences ◽  
Immunofluorescent Staining ◽  
Detectable Effect ◽  
Nih 3T3

Wild-type and mutant chicken integrin beta 1 subunit (beta 1c) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-beta 1c antibodies. Mutant beta 1c integrins containing internal deletions of 13 amino acids adjacent to the membrane, delta 759-771, and 20 centrally located amino acids, delta 771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (797-800). The 11-residue cyto-1 signal is only found on integrin beta subunit sequences, except beta 4. Four residues within this region, D764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an alpha helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conformation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (797-800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.

scholarly journals Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

1994 ◽  
Vol 5 (9) ◽  
pp. 977-988 ◽  
Author(s):  
S Kawaguchi ◽  
J M Bergelson ◽  
R W Finberg ◽  
M E Hemler
Keyword(s):  
Amino Acids ◽  
Cell Adhesion ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Inverse Correlation ◽  
Cytoplasmic Domain ◽  
Negative Regulator ◽  
Wild Type

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.

RPS6 phosphorylation occurs to a greater extent in the periphery of human skeletal muscle fibers, near focal adhesions, after anabolic stimuli

2021 ◽  
Author(s):  
Nathan Hodson ◽  
Michael Mazzulla ◽  
Maksym N. H. Holowaty ◽  
Dinesh Kumbhare ◽  
Daniel R. Moore
Keyword(s):  
Skeletal Muscle ◽  
Focal Adhesion ◽  
Human Skeletal Muscle ◽  
Muscle Fibers ◽  
Focal Adhesions ◽  
Immunofluorescent Staining ◽  
Whole Body ◽  
Vastus Lateralis Muscle ◽  
Cell Periphery ◽  
Rps6 Phosphorylation

Following anabolic stimuli (mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1-mediated phosphorylation events occur in these peripheral regions or prior to translocation (i.e. in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6Ser240/244, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7;23±5yrs;76.8±3.6kg;13.6±3.8%BF, FED) or following a whole-body resistance exercise bout (n=7;22±2yrs;78.1±3.6kg;12.2±4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. RPS6Ser240/244 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that RPS6Ser240/244 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo.

scholarly journals Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK.

1995 ◽  
Vol 182 (4) ◽  
pp. 1089-1099 ◽  
Author(s):  
K Tachibana ◽  
T Sato ◽  
N D'Avirro ◽  
C Morimoto
Keyword(s):  
Amino Acids ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Fusion Proteins ◽  
Focal Adhesions ◽  
Immunohistochemical Analysis ◽  
Binding Activity ◽  
Glutathione S Transferase ◽  
Cytoskeleton Protein ◽  
Direct Association

Focal adhesion kinase (pp125FAK) is localized to focal adhesions and tyrosine phosphorylated by the engagement of beta 1 integrins. However, it is unclear how pp125FAK is linked to integrin molecules. We demonstrate that pp125FAK is directly associated with paxillin, a 68-kD cytoskeleton protein. The COOH-terminal domain of pp125FAK spanning FAK residues 919-1042 is sufficient for paxillin binding and has vinculin-homologous amino acids, which are essential for paxillin binding. Microinjection and subsequent immunohistochemical analysis reveal that glutathione S-transferase-FAK fusion proteins, which bind to paxillin, localize to focal adhesions, whereas fusion proteins with no paxillin-binding activity do not localize to focal adhesions. These findings strongly suggest that pp125FAK is localized to focal adhesions by the direct association with paxillin.

Reversible Association of 14-3-3 with Platelet Glycoprotein Ibα Regulates the Ability of Cells to Adhere, Roll and Spread On VWF

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1061-1061
Author(s):  
Adam D. Munday ◽  
Jose A. Lopez
Keyword(s):  
Amino Acids ◽  
Cho Cells ◽  
Platelet Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Stress Fibers ◽  
Platelet Glycoprotein ◽  
Shear Rates ◽  
Platelet Spreading

Abstract Abstract 1061 Platelet adhesion to sites of vascular injury is required for the arrest of bleeding. Initial platelet adhesion is mediated by binding of von Willebrand factor (VWF) to the platelet glycoprotein (GP) Ib-IX-V complex, leading to the activation of integrin α IIbβ3 and other molecules that mediate firm adhesion, spreading and thrombus formation. The GPIb-IX-V complex comprises 4 polypeptides: GPIbα, GPIbβ, GPIX and GPV, in a 2:4:2:1 stoichiometry. Only the first three polypeptides are required for full VWF binding function. GPIbα is a 610 amino acid polypeptide that binds every known complex ligand within its N-terminal 300 amino acids. The cytoplasmic domain comprises 96 amino acids and contains binding sites for filamin, PI 3-kinase and the scaffolding protein 14-3-3. The association of 14-3-3 with the GPIbα cytoplasmic domain regulates the affinity for VWF. Typically, 14-3-3 requires phosphoserine- or phosphothreonine-containing motifs to bind target proteins. One such motif is in the GPIbα cytoplasmic domain surrounding Ser609, which is phosphorylated and known to bind 14-3-3. Mutation of Ser609 to Ala abrogates 14-3-3 association, which has been proposed to reduce the ability of GPIbα to bind VWF. Platelet aggregation results in the dissociation of 14-3-3 from a subpopulation of GPIbα. Ser609 also becomes dephosphorylated upon platelet spreading. To dissect further the functional roles of 14-3-3 association with GPIbα, we expressed in Chinese hamster ovary (CHO) cells GPIb-IX complexes (GPIbα, GPIbβ, and GPIX) containing either wild type GPIbα, or GPIbα mutants S609A or S609E. In other proteins, mutation of Ser to Glu at the 14-3-3 binding site mimics phosphoserine, recapitulates 14-3-3 binding and often prevents 14-3-3 dissociation. We first assessed the ability of the WT and mutant GPIbα to associate with 14-3-3. As expected, we detected little 14-3-3 binding to GPIbα S609A. GPIbα S609E bound 14-3-3 to the same extent as did WT GPIbα, indicating that the Glu substitution was able to mimic Ser phosphorylation at residue 609. We then assessed the ability of the CHO cells to attach to and roll on VWF under flow over a wide range of shear rates. At 3.26 and 10 dyne/cm2 the α 609A and α 609E cells rolled twice as fast as the WT cells. Both CHO cells and platelets display a characteristic velocity nadir as shear rates increase. The α 609A and α 609E cell showed defective shear-enhanced adhesion; their slowest velocity was ∼3-fold faster than the WT cells. Because GPIbα is dephosphorylated upon platelet spreading, we also assessed the effect of the mutations on cell spreading on VWF. All three cell lines adhered similarly to VWF but a higher percentage of α 609A cells spread (67% vs 58% for WT and α 609E). Of the spread cells, the α S609E cells spread less well; their spread area was 15% less than the WT and α S609A cells. The morphology of the adherent, spread cells was dramatically different among the different cell lines. WT cells displayed a few filopodial extensions along with punctate phalloidin staining indicative of focal adhesions. In some cases the cells displayed stress fibers. The α S609A cells extended more and longer filopodia than the WT cells but had fewer focal adhesions and more stress fibers. The CHO α S609E cells extended thin filopodia that tended to be polarized at two sides of the cell body, and had fewer focal adhesions and no stress fibers. We also examined the effect of the mutations on localization of the GPIb-IX complex to lipid raft membrane microdomains, which is required for platelet adhesion to VWF. Raft GPIbα was reduced by 40% in the S609A cells but increased 1.6-fold in the S609E cells. In summary, lack of 14-3-3 association decreased raft localization of the complex, reduced shear-induced cell adhesion, but increased cell spreading. Stable 14-3-3 association increased raft localization, but decreased shear-induced cell adhesion and decreased the ability of cells to fully spread. Together, our results demonstrate that regulated 14-3-3 association mediated by the phosphorylation status of S609 is required for coordinated adhesion, and cell spreading. Together, our results demonstrate that the functions of the GPIb-IX complex are regulated by the ability of GPIbα Ser609 to both bind and release 14-3-3 and suggest that it is not 14-3-3 binding per se that regulates GPIbα function. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phosphatidylinositol 3,4,5-Trisphosphate Directs Association of Src Homology 2-containing Signaling Proteins with Gelsolin

2001 ◽  
Vol 276 (50) ◽  
pp. 47434-47444 ◽  
Author(s):  
Meenakshi A. Chellaiah ◽  
Rajat S. Biswas ◽  
David Yuen ◽  
Ulises M. Alvarez ◽  
Keith A. Hruska
Keyword(s):  
Bone Resorption ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Protein Interactions ◽  
Lipid Extraction ◽  
Focal Adhesions ◽  
Amino Acid Sequences ◽  
Signaling Proteins ◽  
Src Homology 2 ◽  
Src Homology

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130Cas, focal adhesion kinase, integrin αvβ3, vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, c-Src, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of αvβ3-regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.

NPXY motifs control the recruitment of the alpha5beta1 integrin in focal adhesions independently of the association of talin with the beta1 chain

1997 ◽  
Vol 110 (12) ◽  
pp. 1421-1430 ◽  
Author(s):  
L. Vignoud ◽  
C. Albiges-Rizo ◽  
P. Frachet ◽  
M.R. Block
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Focal Adhesions ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Membrane Receptors ◽  
Sufficient Information ◽  
Protein Protein Interactions ◽  
Exact Function ◽  
Beta1 Subunit

With the exception of the divergent beta4 and beta8 chains, the integrin beta subunit cytoplasmic domains are short and highly conserved sequences. Consensus motifs are found among the different cytoplasmic beta chains. Experiments using chimeric receptors demonstrated that the 47 amino acids of the beta1 subunit cytoplasmic domain contain sufficient information to target integrins to adhesion plaques. Three clusters of amino acids, named cyto-1, cyto-2 and cyto-3, seem to contribute to this localization. Cyto-2 and cyto-3 exhibit NPXY motifs. At present, the exact function of these motifs remains unknown but it is likely that these sequences are involved in protein-protein interactions. Although NPXY motifs often act as internalization signals at the cytoplasmic tail of membrane receptors, our previous results showed that the two NPXY motifs are not responsible for the alpha5beta1 integrin endocytosis. Herein, we address the question of the role of the two highly conserved NPXY motifs found in the beta1 cytoplasmic domain, and which correspond to the conserved domains cyto-2 and cyto-3. We demonstrate that, within the integrin beta1 cytoplasmic tail, the two NPXY motifs are required for the recruitment of the integrin in focal adhesions. In addition, our results indicate that these two motifs control but do not belong to the talin-binding sites. Finally, the analysis of the phenotypes of NPXY mutants reveals that the interaction of talin with the beta1 cytosolic domain is not sufficient to target the integrins to focal adhesions.

Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin

1994 ◽  
Vol 107 (2) ◽  
pp. 709-717 ◽  
Author(s):  
C.K. Wood ◽  
C.E. Turner ◽  
P. Jackson ◽  
D.R. Critchley
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Binding Site ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Terminal Region ◽  
Nih3t3 Cells ◽  
Cell Biol

Paxillin and vinculin are cytoskeletal proteins that colocalise to focal adhesions, specialised regions of the cell involved in attachment to the extracellular matrix. These two molecules form part of a complex of proteins that link the actin network to the plasma membrane. Paxillin has been shown to bind directly in vitro to the C-terminal region of vinculin (Turner et al. (1990). J. Cell Biol. 111, 1059–1068), which also contains a focal adhesion targeting sequence (Bendori et al. (1989). J. Cell Biol. 108, 2383–2393). In the present study, we have used a series of vinculin deletion mutants to map more precisely the sites in vinculin responsible for paxillin binding and focal adhesion localisation. A glutathione-S-transferase fusion protein spanning vinculin residues 881–1000 was sufficient to support 125I-paxillin binding in a gel-blot assay while no detectable binding was observed to a fusion protein spanning residues 881–978. Transfection experiments using cDNAs encoding chick vinculin residues 398–1066 and 398–1028 demonstrated that amino acids C-terminal to residue 1028 were not necessary for targeting to focal adhesions. In contrast, a vinculin polypeptide expressed from a cDNA encoding residues 398–1000 failed to localise to focal adhesions in stably transfected NIH3T3 cells. We have therefore identified a region of 50 amino acids (residues 979–1028) within the C-terminal region of vinculin that contains both the paxillin-binding site and the focal adhesion targeting sequence. This region is highly conserved in human and chicken vinculin and is likely to be important in regulation of the assembly of focal adhesions.

α1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins

2001 ◽  
Vol 356 (1) ◽  
pp. 233-240 ◽  
Author(s):  
Klemens LÖSTER ◽  
Dörte VOSSMEYER ◽  
Werner HOFMANN ◽  
Werner REUTTER ◽  
Kerstin DANKER
Keyword(s):  
Signal Transduction ◽  
Focal Adhesion ◽  
Protein Interactions ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Types ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Adhesion Receptors ◽  
Focal Adhesion Formation

Integrins are heterodimeric adhesion receptors consisting of α- and β-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighbouring cells. These interactions induce various signal transduction pathways in many cell types, leading to cytoskeletal reorganization, phosphorylation and induction of gene expression. Integrin ligation leads to cytoplasmic protein–protein interactions requiring both integrin cytoplasmic domains, and these domains are initiation points for focal adhesion formation and subsequent signal transduction cascades. In previous studies we have shown that the very short cytoplasmic α1 tail is required for post-ligand events, such as cell spreading as well as actin stress-fibre formation. In the present paper we report that cells lacking the cytoplasmic domain of the α1 integrin subunit are unable to form proper focal adhesions and that phosphorylation on tyrosine residues of focal adhesion components is reduced on α1β1-specific substrates. The α1 cytoplasmic sequence is a specific recognition site for focal adhesion components like paxillin, talin, α-actinin and pp125FAK. It seems to account for α1-specific signalling, since when peptides that mimic the cytoplasmic domain of α1 are transferred into cells, they influence α1β1-specific adhesion, presumably by competing for binding partners. For α1 integrin/protein binding, the conserved Lys-Ile-Gly-Phe-Phe-Lys-Arg motif and, in particular, the two lysine residues, are important.

scholarly journals Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component.

1994 ◽  
Vol 5 (2) ◽  
pp. 183-192 ◽  
Author(s):  
A Woods ◽  
J R Couchman
Keyword(s):  
Heparan Sulfate ◽  
Focal Adhesion ◽  
Type I Collagen ◽  
Core Protein ◽  
Mammalian Species ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Type I ◽  
Adhesion Component

Focal adhesion formation in fibroblasts results from complex transmembrane signaling processes initiated by extracellular matrix molecules. Although a role for integrins with attendant tyrosine kinases has been established, there is evidence that cell surface heparan sulfate proteoglycans (HSPGs) are also involved with an associated role of protein kinase C. The identity of the proteoglycan has remained elusive, but we now report that syndecan 4 (ryudocan/amphiglycan) is present in focal adhesions of a number of cell types. Affinity-purified antibodies raised against a unique portion of the cytoplasmic domain of syndecan 4 core protein recognized an HSPG of similar characteristics to those of syndecan 4. These antibodies stained focal adhesions only after cell permeabilization and recognized differing mammalian species. Syndecan 4 was associated with focal adhesions that contained either beta 1 or beta 3 integrin subunits and those that formed on substrates of fibronectin, laminin, vitronectin, or type I collagen. No focal adhesions were found that were vinculin-containing but lacked syndecan 4. In contrast, syndecan 2, whose cytoplasmic domain is closely homologous to syndecan 4, does not appear to be a focal adhesion component. Thus, syndecan 4 represents a new transmembrane focal adhesion component, probably involved in their assembly.

scholarly journals Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding.

1996 ◽  
Vol 135 (4) ◽  
pp. 1109-1123 ◽  
Author(s):  
M C Brown ◽  
J A Perrotta ◽  
C E Turner
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Amino Terminal ◽  
Deletion Mutagenesis ◽  
Lim Domains ◽  
Carboxyl Terminal

Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by &gt;90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.

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