SEARCH · PubMed Health

Results for “Integrin”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources

PubMed2006

Binding of small mono- and oligomeric integrin ligands to membrane-embedded integrins monitored by surface plasmon-enhanced fluorescence spectroscopy.

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.

Amino Acid Sequence↗

PubMed1998

Regulation of integrin function: evidence that bivalent-cation-induced conformational changes lead to the unmasking of ligand-binding sites within integrin alpha5 beta1.

The molecular mechanisms that regulate integrin-ligand binding are unknown; however, bivalent cations are essential for integrin activity. According to recent models of integrin tertiary structure, sites involved in ligand recognition are located on the upper face of the seven-bladed beta-propeller formed by the N-terminal repeats of the alpha subunit and on the von Willebrand factor A-domain-like region of the beta subunit. The epitopes of function-altering monoclonal antibodies (mAbs) cluster in these regions of the alpha and beta subunits; hence these mAbs can be used as probes to detect changes in the exposure or shape of the ligand-binding sites. Bivalent cations were found to alter the apparent affinity of binding of the inhibitory anti-alpha5 mAbs JBS5 and 16, the inhibitory anti-beta1 mAb 13, and the stimulatory anti-beta1 mAb 12G10 to alpha5 beta1. Analysis of the binding of these mAbs to alpha5beta1 over a range of Mn2+, Mg2+ or Ca2+ concentrations demonstrated that there was a concordance between the ability of cations to elicit conformational changes and the ligand-binding potential of alpha5 beta1. Competitive ELISA experiments provided evidence that the domains of the alpha5 and beta1 subunits recognized by mAbs JBS5/16 and 13/12G10 are spatially close, and that the distance between these two domains is increased when alpha5 beta1 is occupied by bivalent cations. Taken together, our findings suggest that bivalent cations induce a conformational relaxation in the integrin that results in exposure of ligand-binding sites, and that these sites lie near an interface between the alpha subunit beta-propeller and the beta subunit putative A-domain.

Antibodies, Monoclonal↗

PubMed1997

Human leukaemic (HMC-1) and normal skin mast cells express beta 2-integrins: characterization of beta 2-integrins and ICAM-1 on HMC-1 cells.

Mast cells are bone marrow-derived, ubiquitous connective tissue resident cells. However, their mechanisms of migration, the distribution of immature and mature cells and their interaction with other inflammatory cells are largely unclarified. Possibly, beta 2-integrins play an important role in these processes. In the present investigation, the authors studied the expression and regulation of the beta 2-integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18) and of the LFA-1/Mac-1 counter-receptor intercellular adhesion molecule-1 (ICAM-1; CD54) on leukaemic (HMC-1 cell subclone 5C6) and on normal mature human skin mast cells. The HMC-1 cells clearly expressed CD11a, CD18 and CD54, while expression of CD11b and CD11c was low. The apparent molecular weights were 180 kDa (CD11a), 95 kDa (CD18) and 90 kDa (CD54) as determined by Western blot analysis. Phorbol myristate acetate (PMA) induced a time- and dose-dependent up-regulation of CD11a, CD11b, CD11c, CD18 and CD54 that was inhibited by cycloheximide, suggesting a dependence on de novo protein synthesis. Enhanced expression of CD11a, CD11b, CD11c and CD18 could also be confirmed at the gene level as demonstrated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Increased expression of LFA-1/ICAM-1 in response to PMA was accompanied by strong enhancement of homotypic cell aggregation, suggesting that newly synthesized LFA-1/ICAM-1 is functionally active. In order to determine a physiologically relevant way of mast cell beta 2-integrin modulation, several cytokines and chemotactic mediators (interleukin-4, IL-4; nerve growth factor beta, NGF beta; C5a; and leukotriene B4, LTB4) were tested for their influence on adhesion molecule cell surface density. Only LTB4 was shown specifically to up-regulate CD11a and CD18, but not CD11b or CD11c. The presence of CD11a, CD11c and CD18 could be confirmed on a low percentage of normal skin mast cells by immunofluorescence, using a double staining technique. In comparison to normal skin, a significantly higher percentage of CD18+ mast cells was found in inflammatory dermatoses such as psoriasis vulgaris, atopic dermatitis and lichen planus. Therefore, mast cell beta 2-integrins possibly play an important role during homing of immature mast cells as well as during the interaction of activated mast cells with other inflammatory cells.

Base Sequence↗

PubMed2001

A membrane-distal segment of the integrin alpha IIb cytoplasmic domain regulates integrin activation.

Previous evidence suggests that interactions between integrin cytoplasmic domains regulate integrin activation. We have constructed and validated recombinant structural mimics of the heterodimeric alpha(IIb)beta(3) cytoplasmic domain. The mimics elicited polyclonal antibodies that recognize a combinatorial epitope(s) formed in mixtures of the alpha(IIb) and beta(3) cytoplasmic domains but not present in either isolated tail. This epitope(s) is present within intact alpha(IIb)beta(3), indicating that interaction between the tails can occur in the native integrin. Furthermore, the combinatorial epitope(s) is also formed by introducing the activation-blocking beta(3)(Y747A) mutation into the beta(3) tail. A membrane-distal heptapeptide sequence in the alpha(IIb) tail ((997)RPPLEED) is responsible for this effect on beta(3). Membrane-permeant palmitoylated peptides, containing this alpha(IIb) sequence, specifically blocked alpha(IIb)beta(3) activation in platelets. Thus, this region of the alpha(IIb) tail causes the beta(3) tail to resemble that of beta(3)(Y747A) and suppresses activation of the integrin.

Amino Acid Sequence↗

PubMed2004

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin alpha 2 beta 1.

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.

Androstadienes↗

PubMed2004

VP7 mediates the interaction of rotaviruses with integrin alphavbeta3 through a novel integrin-binding site.

Rotavirus entry is a complex multistep process that depends on the trypsin cleavage of the virus spike protein VP4 into polypeptides VP5 and VP8 and on the interaction of these polypeptides and of VP7, the second viral surface protein, with several cell surface molecules, including integrin alphavbeta3. We characterized the effect of the trypsin cleavage of VP4 on the binding to MA104 cells of the sialic acid-dependent virus strain RRV and its sialic acid-independent variant, nar3. We found that, although the trypsin treatment did not affect the attachment of these viruses to the cell surface, their binding was qualitatively different. In contrast to the trypsin-treated viruses, which initially bound to the cell surface through VP4, the non-trypsin-treated variant nar3 bound to the cell through VP7. Amino acid sequence comparison of the surface proteins of rotavirus and hantavirus, both of which interact with integrin alphavbeta3 in an RGD-independent manner, identified a region shared by rotavirus VP7 and hantavirus G1G2 protein in which six of nine amino acids are identical. This region, which is highly conserved among the VP7 proteins of different rotavirus strains, mediates the binding of rotaviruses to integrin alphavbeta3 and probably represents a novel binding motif for this integrin.

Amino Acid Sequence↗

PubMed1997

Integrin-mediated regulation of hematopoiesis: do BCR/ABL-induced defects in integrin function underlie the abnormal circulation and proliferation of CML progenitors?

Hematopoiesis takes place in close contact with the marrow microenvironment. Normal progenitors adhere through a variety of receptors to stroma and extracellular matrix components, including fibronectin. Adhesion through integrins to fibronectin may not only serve to anchor progenitors to the microenvironment but also to directly alter the proliferative behavior of normal hematopoietic progenitors. Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell. At the molecular level, CML is characterized by the BCR/ABL gene rearrangement which encodes for the oncoprotein, p210bcr-abl. Presence of the p210bcr-abl tyrosine kinase is necessary and sufficient for the malignant transformation of hematopoietic cells. Clinically, CML is characterized by an abnormal, premature release of primitive progenitors and precursors in the blood and by the continuous proliferation of the malignant progenitor population. In vitro, CML progenitors fail to adhere to or be regulated by marrow stroma. Since CML progenitors express similar numbers of integrin adhesion receptors as normal progenitors, functional rather than quantitative differences of these receptors on CML progenitors may be responsible for the abnormal circulation and proliferation of the malignant clone. In this manuscript we will review the role of integrin adhesion receptors present on normal hematopoietic progenitors in the regulation of their proliferation and discuss signal transduction mechanisms that may be responsible for these effects. We will also discuss the integrin defect in CML which may be caused by the presence of the oncoprotein, P210bcr-abl, and may explain the abnormal trafficking and proliferation observed in CML.

Bone Marrow↗

PubMed2005

The integrins of the urochordate Ciona intestinalis provide novel insights into the molecular evolution of the vertebrate integrin family.

BACKGROUND: Integrins are a functionally significant family of metazoan cell surface adhesion receptors. The receptors are dimers composed of an alpha and a beta chain. Vertebrate genomes encode an expanded set of integrin alpha and beta chains in comparison with protostomes such as drosophila or the nematode worm. The publication of the genome of a basal chordate, Ciona intestinalis, provides a unique opportunity to gain further insight into how and when the expanded integrin supergene family found in vertebrates evolved. RESULTS: The Ciona genome encodes eleven alpha and five beta chain genes that are highly homologous to their vertebrate homologues. Eight of the alpha chains contain an A-domain that lacks the short alpha helical region present in the collagen-binding vertebrate alpha chains. Phylogenetic analyses indicate the eight A-domain containing alpha chains cluster to form an ascidian-specific clade that is related to but, distinct from, the vertebrate A-domain clade. Two Ciona alpha chains cluster in laminin-binding clade and the remaining chain clusters in the clade that binds the RGD tripeptide sequence. Of the five Ciona beta chains, three form an ascidian-specific clade, one clusters in the vertebrate beta1 clade and the remaining Ciona chain is the orthologue of the vertebrate beta4 chain. CONCLUSION: The Ciona repertoire of integrin genes provides new insight into the basic set of these receptors available at the beginning of vertebrate evolution. The ascidian and vertebrate alpha chain A-domain clades originated from a common precursor but radiated separately in each lineage. It would appear that the acquisition of collagen binding capabilities occurred in the chordate lineage after the divergence of ascidians.

Amino Acid Sequence↗

PubMed1995

An alternatively spliced exon in the extracellular domain of the human alpha 6 integrin subunit--functional analysis of the alpha 6 integrin variants.

Variants in the extracellular domain of the integrin alpha 7 subunit which arise as a consequence of alternative splicing of mRNA have recently been reported. Two alternative exons, X1 and X2, have been identified in the alpha 7 gene, and homologous exons were found for alpha 6 (Ziober et al., 1993). In this study, we have isolated the region of the alpha 6 gene containing exons X1 and X2 that are, like those of alpha 7, located between stretches of DNA that encode the homologous repeat domains III and IV, proximal to the three divalent cation binding sites of the alpha 6 subunit. We demonstrated by reverse transcriptase polymerase chain reactions and confirmed by sequencing that alpha 6X1 and alpha 6X1X2 mRNAs are generated by alternative splicing of exon X2. The alpha 6X1X2 mRNA is expressed in a limited number of tissues and cell lines and it is always co-expressed with the ubiquitous alpha 6X1 mRNA. Stable transfection of K562 cells with full length cDNAs for the alpha 6AX1X2 and beta 4 subunits resulted in cell populations that expressed the alpha 6AX1X2 variant, in association with either beta 1 or beta 4, on their surface. In addition, a population of cells was isolated that expressed the alpha 6AX1X2 variant at low levels and almost exclusively in association with beta 1. Comparison of the alpha 6AX1X2 integrins with alpha 6AX1 using similarly transfected cells showed no obvious differences between the alternative extracellular alpha 6A isoforms with respect to ligand specificity and activation-dependency of ligand binding. After treatment with the anti-beta 1 stimulatory antibody TS2/16, both the alpha 6AX1 beta 1 and alpha 6AX1X2 beta 1 integrin variants mediated cell adhesion to EHS tumor laminin (laminin-1), kalinin (laminin-5), human placental (laminin-2 and -4) and bovine kidney laminins. In contrast, the alpha 6AX1 beta 4 and alpha 6AX1X2 beta 4 integrins also mediated cell adhesion to laminin and kalinin without stimulation. Furthermore, the different transfectants did not differ in their ability to spread on kalinin. The presented data indicate that the X2 region in alpha 6 is not involved in defining ligand specificity or affinity.

Alternative Splicing↗

PubMed1997

Identification of the cleavage sites in the alpha6A integrin subunit: structural requirements for cleavage and functional analysis of the uncleaved alpha6Abeta1 integrin.

The alpha6A and alpha6B integrin subunits are proteolytically cleaved during biosynthesis into a heavy chain (120 kDa) that is disulphide-linked to one of two light chains (31 or 30 kDa). Analysis of the structure of the alpha6A subunit on the carcinoma cell line T24 and human platelets demonstrated that the two light chains of alpha6 are not differentially glycosylated products of one polypeptide. Rather they possess different polypeptide backbones, which presumably result from proteolytic cleavage at distinct sites in the alpha6 precursor. Mutations were introduced in the codons for the R876KKR879, E883K884, R890K891 and R898K899 sequences, the potential proteolytic cleavage sites, and wild-type and mutant alpha6A cDNAs were transfected into K562 cells. The mutant alpha6A integrin subunits were expressed in association with endogenous beta1 at levels comparable to that of wild-type alpha6Abeta1. A single alpha6 polypeptide chain (150 kDa) was precipitated from transfectants expressing alpha6A with mutations or deletions in the RKKR sequence. Mutations in the EK sequence yielded alpha6A subunits that were cleaved once into a heavy and a light chain, whereas alpha6A subunits with mutations in one of the two RK sequences were, like wild-type alpha6A, cleaved into one heavy and two light chains. Thus a change in the RKKR sequence prevents the cleavage of alpha6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing of the N-termini of the two alpha6A light chains from platelets demonstrated that cleavage occurs after Arg879 and Lys884. Because alpha6(RKKG), alpha6(GKKR) and alpha6(RGGR) subunits were not cleaved it seems that both the arginine residues and the lysine residues are essential for cleavage of RKKR. alpha6A mutants with the RKKR sequence shifted to the EK site, in such a way that the position of the arginine residue after which cleavage occurs corresponds exactly to Lys884, were partly cleaved, whereas alpha6A mutants with the RKKR sequence shifted to other positions in the alpha6A subunit, including one in which it was shifted two residues farther than the EK cleavage site, were not cleaved. In addition, alpha6A mutants with an alpha5-like cleavage site, i.e. arginine, lysine and histidine residues at positions -1, -2 and -6, were not cleaved. Thus both an intact RKKR sequence and its proper position are essential. After activation by the anti-beta1 stimulatory monoclonal antibody TS2/16, both cleaved and uncleaved alpha6Abeta1 integrins bound to laminin-1. The phorbol ester PMA, which activates cleaved wild-type and mutant alpha6Abeta1, did not activate uncleaved alpha6Abeta1. Thus uncleaved alpha6Abeta1 is capable of ligand binding, but not of inside-out signalling. Our results suggest that cleavage of alpha6 is required to generate a proper conformation that enables the affinity modulation of the alpha6Abeta1 receptor by PMA.

Amino Acid Sequence↗

PubMed1996

Inducible tyrosine phosphorylation of the beta3 integrin requires the alphaV integrin cytoplasmic tail.

We have found that the integrin beta3 chain can be phosphorylated on tyrosine residues in K562 cells transfected with alphavbeta3. Tyrosine phosphorylation of the beta3 cytoplasmic tail is induced by adhesion to alphavbeta3-specific ligand or antibody or by incubation in manganese-containing buffer. Under the same conditions, beta5 does not become tyrosine-phosphorylated in K562 transfected with alphavbeta5. Phosphorylation of the beta3 subunit requires the simultaneous presence of the alphav subunit cytoplasmic tail, because neither the alphaIIb subunit nor a truncated alphav subunit is sufficient to permit phosphorylation of beta3 when coexpressed as a heterodimer with beta3. Finally, tyrosine phosphorylation of the beta3 cytoplasmic tail occurs on both human and murine beta3 and is inducible in the ovarian carcinoma OV10 as well, independent of expression of integrin-associated protein (CD47). Tyrosine phosphorylation of the beta3 integrin subunit facilitates association of Grb-2, an adaptor protein leading to activation of the Ras signaling pathway, and may contribute to the unique functional and signaling capabilities of alphavbeta3.

Adaptor Proteins, Signal Transducing↗

PubMed1999

CD9 is expressed in extravillous trophoblasts in association with integrin alpha3 and integrin alpha5.

The CD9 molecule is a 24-27 kDa cell surface glycoprotein, which may be related to Schwann cell migration and adhesion. In this study, we examined the expression of CD9 in human extravillous trophoblasts, which invade into the endometrium during implantation and placentation. CD9 was detected immunohistochemically on the extravillous trophoblasts in the cell columns of first trimester placentae, but not on villous trophoblasts. In the second and third trimester, CD9 was highly expressed on the extravillous trophoblasts in the basal plate of placentae, and in the chorion laeve in the fetal membrane of term placentae. The molecular mass of CD9 in the chorion laeve was shown to be 27 kDa by Western blotting. The mRNA of CD9 was also detected in the chorion laeve by reverse transcription-polymerase chain reaction (RT-PCR). Proteins were purified from chorion laeve by affinity chromatography with anti-integrin alpha3 and alpha5 monoclonal antibodies and Western blotting, revealed that CD9 was associated with both integrins. These findings indicate that CD9 is a differentiation-related molecule present in the extravillous trophoblasts. Since it is associated with integrin alpha5 which has been proposed to regulate trophoblast invasion, CD9 may be implicated in trophoblast invasion at the feto-maternal interface.

Antigens, CD↗

PubMed2003

Genetic evidence for convergence of c-Kit- and alpha4 integrin-mediated signals on class IA PI-3kinase and the Rac pathway in regulating integrin-directed migration in mast cells.

Mast cells play a critical role in host defense against a number of pathogens. Increased mast cell infiltration has been described in allergic asthma, in rheumatoid arthritis, and during helminthes infection. Despite the importance of mast cells in allergic disease and defense against infection, little is known about the mechanisms by which mast cells migrate to various tissues under steady state conditions or during infection or inflammation. Here, we show that activation of c-Kit by its ligand, stem cell factor (SCF), cooperates with alpha4 integrin in inducing directed migration of mast cells on fibronectin. A reduction in migration and activation of a small G protein, Rac, was observed in mast cells derived from class IA phosphoinositide-3 kinase (PI-3kinase)-deficient mice in response to SCF stimulation and in mast cells expressing the dominant-negative Rac (RacN17), as well as in mast cells deficient in the hematopoietic-specific small G protein, Rac2. In addition, a PI-3kinase inhibitor inhibited alpha4- as well as SCF-induced migration in a dose-dependent fashion. In contrast, a mitogen-activated protein kinase (MAPK) inhibitor had little effect. Consistent with the pharmacologic results, abrogating the binding of the p85alpha subunit of class IA PI-3kinase to c-Kit also resulted in inhibition of SCF-induced migration on fibronectin. These genetic and biochemical data demonstrate that both c-Kit and alpha4 integrin signaling are linked to class IA PI-3kinase and Rac pathways and regulate integrin-directed (haptotactic) migration in mast cells.

Animals↗

PubMed2003

Thrombospondin-bound integrin-associated protein (CD47) physically and functionally modifies integrin alphaIIbbeta3 by its extracellular domain.

Integrin-associated protein (IAP/CD47) is a receptor for the C-terminal cell binding domain of thrombospondin (TS). A peptide from the C-terminal cell binding domain, KRFYVVMWKK (4N1K) binds to IAP and stimulates the integrin-dependent cell functions, including platelet aggregation. We investigated the mechanism by which TS-bound IAP modulates the affinity of platelet integrin, alphaIIbbeta3. Platelet aggregation induced by 4N1K was not completely inhibited by energy depletion with sodium azide and 2-deoxy-d-glucose, although ADP or collagen-induced platelet response was completely inhibited. The binding of ligand-mimetic antibody PAC1 to alphaIIbbeta3 was also induced in the energy-depleted platelets. In the transfected Namalwa cells, 4N1K induced activation of the alphaIIbbeta3 with mutated beta3 (Ser-752 to Pro), which is a non-responsive form to inside-out signaling, as well as wild type alphaIIbbeta3. The truncated form of IAP with only the extracellular immunoglobulin-like (Ig) domain was sufficient for the activation of alphaIIbbeta3 in Chinese hamster ovary cells, although the IAP-mediated intracellular signaling was abolished, which was monitored by the absence of down-regulation of mitogen-activated protein kinase phosphorylation. Furthermore, the soluble recombinant Ig domain of IAP induced PAC1 binding to alphaIIbbeta3 on Chinese hamster ovary cells when added with 4N1K. Physical association between the soluble recombinant Ig domain of IAP and purified alphaIIbbeta3 was detected in the presence of 4N1K. These data indicate that the extracellular Ig domain of IAP, when bound to TS, interacts with alphaIIbbeta3 and can change alphaIIbbeta3 in a high affinity state without the requirement of intracellular signaling. This extracellular event would be a novel mechanism of affinity modulation of integrin.

Amino Acid Sequence↗

PubMed2003

GPI-80, a beta2 integrin associated glycosylphosphatidylinositol-anchored protein, concentrates on pseudopodia without association with beta2 integrin during neutrophil migration.

Previously, we identified a glycosylphosphatidylinositol (GPI)-anchored protein, designated GPI-80, present on human neutrophils and monocytes. GPI-80 is physically associated with beta2 integrin on the surface of human neutrophils and may be a regulator of neutrophil adherence and migration. However, it is not yet known how GPI-80 regulates cell adhesion and migration. To investigate the physiological role(s) of GPI-80, we examined the topological relationship of GPI-80 and the beta2 integrin subunit (CD18) on resting and migrating human neutrophils by confocal laser microscopy. On resting neutrophils, GPI-80 was evenly distributed on the cell surface and was associated with CD18. On the other hand, during the early phase of migration (5 - 30 minutes), GPI-80 was detected on cell bodies and also on pseudopodia, but CD18 was detected only on cell bodies, where it was associated with GPI-80. In the late phase of migration (60 minutes), GPI-80 was detected only on pseudopodia and its association with CD18 was hardly observed. Furthermore, some of the GPI-80 on pseudopodia of migrating neutrophils during the late phase was associated with urokinase-type plasminogen activator receptor (uPAR), a regulator of beta2 integrin-dependent adherence and migration. The distribution of GPI-80 on cell surfaces is similar to that of uPAR. These observations suggest that GPI-80 belongs to the beta2 integrin-associated GPI-anchored protein family, which has regulatory activity in cell adherence.

Amidohydrolases↗

PubMed1999

Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats.

Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. In this study, we show that ILK is a constituent of cell-matrix focal adhesions. ILK was recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK was absent in E-cadherin-mediated cell-cell adherens junctions. In previous studies, we have identified PINCH, a protein consisting of five LIM domains, as an ILK binding protein. We demonstrate in this study that the ILK-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of ILK and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The ILK ANK repeats domain, which is capable of interacting with PINCH in vitro, could also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex was markedly reduced in the absence of the C-terminal domain of ILK. The PINCH binding defective ANK1 deletion ILK mutant, unlike the wild-type ILK, was unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of ILK. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of ILK, as an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site failed to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where ILK functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating ILK cellular function.

3T3 Cells↗

PubMed1996

Detection of post-translational sulfation of alpha-5 beta-1 integrin and its role in integrin-fibronectin binding.

Fibronectins are glycoproteins of the extracellular matrix composed of two 220-kDa polypeptide chains named A and B bound by two disulfide bridges. Both chains when digested with proteolytic enzymes give rise to six different domains named I to VI that are involved in the ligand properties of this molecule. Fibronectins bind fibrin, collagen, glycosaminoglycan residues and several integrins. In this study, using metabolic radiolabeling of alpha 5 beta 1 integrin with sodium sulfate, an immunoprecipitation reaction, inhibition of sulfate incorporation and a fibronectin-binding assay, we were able to detect this integrin as a sulfated molecule and this sulfation appears to regulate the integrin-fibronectin binding.

Collagen↗

PubMed1995

Mechanisms of VCAM-1 and fibronectin binding to integrin alpha 4 beta 1: implications for integrin function and rational drug design.

Integrin alpha 4 beta 1 can mediate both cell-cell and cell-extracellular matrix adhesion by binding to either fibronectin or vascular cell adhesion molecule 1 (VCAM-1). Both interactions are important for extravasation of leukocytes from the blood implying that rationally designed inhibitors of alpha 4 beta 1 function may be useful for treating a various inflammatory conditions. The mechanisms of ligand binding by alpha 4 beta 1 are complicated by the fact that alternative splicing can generate different isoforms of the receptor-binding domains in both fibronectin and VCAM-1. Therefore, in addition to developing alpha 4 beta 1 antagonists, we have also been interested in identifying isoform-specific functions. Recombinant ligand variants have been tested in adhesion and direct receptor-binding assays and each molecule was found to have a different inherent affinity for alpha 4 beta 1 that endows them with different adhesive activities. This suggests that alternative splicing may regulate alpha 4 beta 1-dependent motility in vivo. The initial strategy that we have adopted to develop alpha 4 beta 1 inhibitors has been to identify key amino acid residues and peptide sequences participating in the receptor-ligand binding event and to use this information to generate synthetic mimetics. Three active sites have been identified in fibronectin by testing truncated proteins, expressing recombinant fragments and screening synthetic peptides. Two of these sites employ versions of a novel integrin-binding motif, LDVP/IDAP. A key active site in VCAM-1 has been identified by similar approaches as the related sequence IDSP. Since IDSP-like sequences are probably used by other integrin-binding immunoglobulins, derivatives of these peptides may turn out to be the forerunners of a new generation of therapeutic agents with multiple applications.

Amino Acid Sequence↗