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J Ylänne

Publications and source records attributed to J Ylänne.

At least 19 recordsLinked to original sources
PubMed2003

Involvement of the beta3 E749ATSTFTN756 region in stabilizing integrin alphaIIbbeta3-ligand interaction.

Platelet integrin alphaIIbbeta3 must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface-bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the beta-subunit plays a central role. We introduced peptides homologous to the E749ATSTFTN756 domain (E-N peptide) and the T755NITYRGT762 domain (T-T peptide) of beta3 in streptolysin O-permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC-1 after stimulation with thrombin. E-N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E-N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E-N peptide did not disturb the binding of PAC-1, which is known to reflect activation of the integrin. E-N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on alphaIIbbeta3. T-T peptide did not affect these processes. In a model for outside-in integrin activation, E-N peptide disrupted the binding of CHO cells expressing alphaIIbbeta3 to surface-bound ligand. Again, T-T peptide had no effect. We conclude that the E749ATSTFTN756 region of the beta3-tail stabilizes the binding of soluble and surface-bound ligand to integrin alphaIIbbeta3 via a mechanism that involves the phosphorylation of FAK.

Animals↗
PubMed2001

Crystal structure of the alpha-actinin rod reveals an extensive torsional twist.

BACKGROUND: Alpha-actinin is a ubiquitously expressed protein found in numerous actin structures. It consists of an N-terminal actin binding domain, a central rod domain, and a C-terminal domain and functions as a homodimer to cross-link actin filaments. The rod domain determines the distance between cross-linked actin filaments and also serves as an interaction site for several cytoskeletal and signaling proteins. RESULTS: We report here the crystal structure of the alpha-actinin rod. The structure is a twisted antiparallel dimer that contains a conserved acidic surface. CONCLUSIONS: The novel features revealed by the structure allow prediction of the orientation of parallel and antiparallel cross-linked actin filaments in relation to alpha-actinin. The conserved acidic surface is a possible interaction site for several cytoplasmic tails of transmembrane proteins involved in the recruitment of alpha-actinin to the plasma membrane.

Actinin↗
PubMed2001

The NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform.

Beta3 integrin adhesion molecules play important roles in wound repair and the regulation of vascular development and three beta3 integrin isoforms (beta3-A, -B, -C) have been described so far. Surface expression of beta3 integrins is dynamically regulated through internalization of beta3 integrins, however, the molecular mechanisms are understood incompletely. To evaluate the role of the cytoplasmic domain of beta3 integrins for internalization, we have generated single chain chimeras with variant and mutated forms of beta3 cytoplasmic domains. Upon transient transfection into chinese hamster ovary cells, it was found that the beta3-A chimera had strongly reduced cell surface expression compared with the corresponding beta3-B, or beta3-C fusion proteins, or the tail-less constructs, whereas steady state levels of all chimeras were near identical. Studies employing cytoplasmic domain mutants showed that the NITY motif at beta3-A 756-759 is critical for plasma membrane expression of beta3-A. Furthermore, delivery of beta3-A to the cell surface was specifically modulated by the cytoplasmic protein beta3-endonexin, a previously described intracellular protein. Coexpression of the native, long form of beta3-endonexin, which does not interact with the beta3 tail, acted as a dominant negative inhibitor of beta3-A-internalization and enhanced steady-state surface expression of the beta3-A-chimera. Furthermore, anti-beta3 antibody-induced internalization of the native beta3 integrin (alpha(IIb)beta3 was dramatically reduced for the Tyr(759)-Ala substitution mutant (alpha(IIb)beta3) (Y759A) and expression of the long isoform of beta3-endonexin substantially decreased the internalization of wild-type alpha(IIb)beta3. Thus, the NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform and beta3-endonexin appears to couple the beta3-A isoform to a specific receptor-recycling pathway.

Amino Acid Sequence↗
PubMed1999

Effects of mutations in the cytoplasmic domain of integrin beta(1) to talin binding and cell spreading.

Integrins are transmembrane proteins linking the extracellular matrix or certain cell-cell contacts to the cytoskeleton. To study integrin-cytoskeleton interactions we wanted to relate talin-integrin interaction to integrin function in cell spreading and formation of focal adhesions. For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin beta(1) (GST-cytobeta(1)) expressed in bacteria. For functional studies chimeric integrins containing the extracellular and transmembrane parts of beta(3) linked to the cytoplasmic domain of beta(1) were expressed in CHO cells as a dimer with the alpha(IIb) subunit. Point mutations in the amino acid sequence N(785)PIY(788) of beta(1) disrupted both the integrin-talin interaction and the ability of the integrin to mediate cell spreading. COOH-terminal truncation of beta(1) at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792). A synthetic peptide from this region of beta(1) (W(780)DTGENPIYKSAV(792)) bound to purified talin and inhibited talin binding to GST-cytobeta(1). The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading. These results confirm the previous finding that a talin-binding site in the integrin beta(1) tail resides at or close to the central NPXY motif and suggest that the integrin-talin interaction is necessary but not sufficient for integrin-mediated cell spreading.

3T3 Cells↗
PubMed1998

Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (alphaIIbbeta3) inhibitors.

The blockade of platelet integrin glycoprotein (GP) IIb/IIIa is a promising new antiplatelet strategy. The binding of ligands or of the ligand-mimetic peptide RGD causes a conformational change of GP IIb/IIIa from the nonactivated to the activated state. Because several blocking agents/inhibitors are ligand-mimetics, the current study evaluates whether these agents have the intrinsic property to activate GP IIb/IIIa. Fibrinogen binding to GP IIb/IIIa on platelets or on CHO cells expressing recombinant GP IIb/IIIa was evaluated by flow cytometry or 125I-labeled fibrinogen. Incubation with the monoclonal antibody (MoAb) fragment c7E3 (abciximab) results in fibrinogen binding to GP IIb/IIIa and in the access of ligand-induced binding sites. At low concentrations (0.01 to 0.1 microgram/mL), this intrinsic activating property of c7E3 can result in platelet aggregation. The disintegrin flavorodin and the RGD analogue fradafiban also induce fibrinogen binding, whereas the blocking MoAbs 2G12 and P2 and the activation-specific MoAb PAC-1 do not. Aspirin and indomethacin cannot block c7E3-induced fibrinogen binding to GP IIb/IIIa, but can inhibit c7E3-induced platelet aggregation. Thus, we conclude that GP IIb/IIIa inhibitors can demonstrate an intrinsic activating property, which can result in fibrinogen binding to GP IIb/IIIa and consequently in platelet aggregation. Cyclooxygenase inhibitors can inhibit platelet aggregation caused by GP IIb/IIIa inhibitors. Further studies will have to evaluate the clinical relevance of the potential intrinsic activating property of GP IIb/IIIa inhibitors and define consequences for the future drug development and evaluation of these potent antiplatelet agents.

Abciximab↗
PubMed1998

Conserved functions of the cytoplasmic domains of integrin beta subunits.

In this article I will discuss the notion that many integrins have similar functions in cell spreading, organization of the cytoskeleton and intracellular signalling. Most of these functions are transmitted through the cytoplasmic domains of integrin beta-subunits. These parts are also quite conserved between most integrins. I will discuss, what is known about the molecules binding to these parts of integrins, and which of those transmit the conserved functions.

Amino Acid Sequence↗
PubMed1996

Expression of megakaryocytic and erythroid properties in human leukemic cells.

Previous studies have suggested that megakaryocytes and erythrocytes may share a common precursor cell. However, studies on the commitment to erythroid and megakaryocytic lineages have been hampered by the lack of suitable human leukemic cell lines having this kind of bipotential differentiation capability. We investigated the coexpression of megakaryocytic and erythroid markers in human leukemic cell lines as well as the capability of these cells to further differentiate upon exposure to differentiation-inducing agents. We report that the JK-1 cell line, previously characterized as a typically erythroid cell line with spontaneous differentiation to red cells, actually coexpressed megakaryocytic cell surface antigens and erythroid spectrins. We also report that the JK-1 cells could be induced to differentiate along the megakaryocytic lineage by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The other cell lines studied variably expressed megakaryocytic and erythroid antigens, the DAMI and CMK cells predominantly megakaryocytic properties and the T-33 and K562 cells some erythroid markers, whereas the HEL cells expressed markers for both lineages of differentiation. Our results suggest that the JK-1 cell line represents an immature cell population that has not yet been committed to either of the two lineages of differentiation. The JK-1 cell line might provide a useful tool for further studies on the transcriptional regulation of erythroid and megakaryocytic phenotypes and for studies on the commitment to these lineages of differentiation. Our results also suggest that the leukemic cell lines show a considerable plasticity in the expression of properties normally specific for distinct lineages of differentiation.

Antigens, CD↗
PubMed1995

The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand binding affinity.

Integrin affinities for ligands can change markedly via a process termed inside-out signaling. We expressed several truncations of the beta 3 cytoplasmic domain in conjunction with an "activating" alpha subunit chimera, alpha IIb alpha 6B. Deletion of the 4 C-terminal residues of the beta 2 tail blocked inside-out signaling as assessed by the binding of an activation-specific antibody, PAC1. Several additional truncations remained in the low affinity state, but complete truncation (beta 3 delta 717) caused PAC1 binding. Activation by this truncation mutant did not depend on the alpha subunit cytoplasmic domain and was resistant to inhibitors of cellular metabolism and the over-expression of an isolated beta 3 cytoplasmic domain. Since deletion of beta 3(Leu717-Asp723) results in a constitutively activated integrin, this membrane-proximal seven amino acids of the beta 3 cytoplasmic domain is required to maintain alpha IIb beta 3 in a default low affinity state. The amino acid sequence of this region is conserved among integrins. Moreover, the conserved membrane-proximal sequence in alpha subunit tails seems to serve a similar function. Consequently, the conserved membrane-proximal regions of both integrin cytoplasmic domains control the ligand binding affinity of the extracellular domain.

Amino Acid Sequence↗
PubMed1995

Mutation of the cytoplasmic domain of the integrin beta 3 subunit. Differential effects on cell spreading, recruitment to adhesion plaques, endocytosis, and phagocytosis.

The cytoplasmic domain of the beta subunit of the alpha IIb beta 3 integrin is required for cell spreading on fibrinogen. Here we report that deletion of six amino acids from the COOH terminus of the beta 3 (I757TYRGT) totally abolished cell spreading and formation of adhesion plaques, whereas retaining Ile757 partially preserved these functions. We further found that substitution of Tyr747 with Ala also abolished alpha IIb beta a-mediated cell spreading. The effects of these and other mutations on additional functions of alpha IIb beta 3 were also studied. Progressive truncations of beta 3, in which stop codons were inserted at amino acid positions 759-756, caused partial defects in the recruitment of alpha IIb beta 3 to preestablished adhesion plaques and a gradual decrease in the ability of alpha IIb beta 3 to mediate internalization of fibrinogen-coated particles. The Tyr747-->Ala substitution mutant was almost totally inactive in both of these assays. Point mutations at Tyr759, and at a conserved area close to the transmembrane domain of beta 3, decreased integrin recruitment to preestablished adhesion plaques but allowed alpha IIb beta 3-mediated formation of these structures and partial cell spreading. Deletion of the cytoplasmic domain of beta 3 did not affect the constitutive endocytosis of alpha IIb beta 3.

Amino Acid Sequence↗
PubMed1994

A point mutation in the integrin beta 3 cytoplasmic domain (S752-->P) impairs bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-IIIa).

Agonist-induced inside-out signaling results in an increased affinity of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) for soluble ligands (fibrinogen [Fg] and PAC1). Ligand binding to integrins initiates outside-in signaling that leads to cellular responses such as cell spreading and focal adhesion formation. A point mutation in the beta 3 cytoplasmic domain (S752-->P) is associated with blocked inside-out alpha IIb beta 3 signaling in a variant Glanzmann's thrombasthenia. This mutation was introduced into beta 3 and cotransfected into Chinese hamster ovary cells with a chimeric alpha subunit consisting of the alpha IIb extracellular and transmembrane domains and the alpha 6B cytoplasmic domain. The substitution of the alpha IIb cytoplasmic domain with that of alpha 6 led to activation of alpha IIb beta 3 to bind PAC1, mimicking inside-out signaling. This effect was reversed by the S752-->P mutation, indicating a disruption of inside-out signaling by the mutation. In addition, transfectants expressing this beta 3 variant showed reduced alpha IIb beta 3-mediated cell spreading on immobilized Fg, focal adhesion, and fibrin clot retraction, suggesting an impairment in outside-in alpha IIb beta 3 signaling. Therefore, a single point mutation in the beta 3 cytoplasmic domain impaired bidirectional signaling through alpha IIb beta 3.

Animals↗
PubMed1993

The effect of differentiation inducers on the integrin expression of K562 erythroleukemia cells.

We studied the effects of differentiation-inducers on the integrin profile and adhesive properties of K562 leukemia cells. The fibronectin (Fn) receptor integrin, alpha 5 beta 1, was the only integrin expressed in suspension cultured K562 cells. When the cells were exposed to 12-0-tetradecanoylphorbol-13-acetate (TPA) immunoreactivity for the beta 1 integrin subunit was slightly enhanced. TPA exposure also induced the appearance of the alpha 2, alpha 3, alpha v and beta 3 integrin subunits, but the platelet integrin subunit alpha IIb was not detected. On the other hand, hemin chloride-induced erythroid differentiation of K562 cells diminished the expression of the alpha 5 beta 1 integrin on the surface of the cells. Adhesion experiments with TPA-exposed K562 cells indicated that although the adherence to the extracellular matrix (ECM) proteins as a rule was low a few cells spread on these proteins. The present results specify the effects of differentiation inducers on the integrin profile of K562 cells and excludes the comprehension that TPA would induce expression of the platelet integrin alpha IIb on their surface. Our results also show, that an increased expression of a certain integrin does not necessarily lead to a comparable adhesion ability on its ligand in vitro.

Cell Adhesion↗
PubMed1993

Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions.

Integrin-mediated cell adhesion often results in cell spreading and the formation of focal adhesions. We exploited the capacity of recombinant human alpha IIb beta 3 integrin to endow heterologous cells with the ability to adhere and spread on fibrinogen to study the role of integrin cytoplasmic domains in initiation of cell spreading and focal adhesions. The same constructs were also used to analyze the role of the cytoplasmic domains in maintenance of the fidelity of the integrin repertoire at focal adhesions. Truncation mutants of the cytoplasmic domain of alpha IIb did not interfere with the ability of alpha IIb beta 3 to initiate cell spreading and form focal adhesions. Nevertheless, deletion of the alpha IIb cytoplasmic domain allowed indiscriminate recruitment of alpha IIb beta 3 to focal adhesions formed by other integrins. Truncation of the beta 3 subunit cytoplasmic domain abolished cell spreading mediated by alpha IIb beta 3 and also abrogated recruitment of alpha IIb beta 3 to focal adhesions. This truncation also dramatically impaired the ability of alpha IIb beta 3 to mediate the contraction of fibrin gels. In contrast, the beta 3 subunit cytoplasmic truncation did not reduce the fibrinogen binding affinity of alpha IIb beta 3. Thus, the integrin beta 3 subunit cytoplasmic domain is necessary and sufficient for initiation of cell spreading and focal adhesion formation. Further, the beta 3 cytoplasmic domain is required for the transmission of intracellular contractile forces to fibrin gels. The alpha subunit cytoplasmic domain maintains the fidelity of recruitment of the integrins to focal adhesions and thus regulates their repertoire of integrins.

Amino Acid Sequence↗
PubMed1992

A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques.

A point mutation in a highly conserved region of the beta 1 subunit, Asp130 to Ala (D130A) substitution, abrogates the Arg-Gly-Asp (RGD)-dependent binding of alpha 5 beta 1 to fibronectin (FN) without disrupting gross structure or heterodimer assembly. The D130A mutation also interferes with binding to invasin, a ligand that lacks RGD sequence. In spite of the lack of detectable FN binding by alpha 5 beta 1(D130A), it was recruited to adhesion plaques formed on FN by endogenous hamster receptors. Thus, intact ligand binding function is not required for recruitment of alpha 5 beta 1 to adhesion plaques. Overexpression of beta 1(D130A) partially interfered with endogenous alpha 5 beta 1 function, thus defining a dominant negative beta 1 integrin mutation.

Adhesins, Bacterial↗
PubMed1992

Integrin distributions in renal cell carcinomas of various grades of malignancy.

We studied 41 renal cell carcinomas, classified according to histologic grades G1 through G3, by indirect immunofluorescence microscopy using a panel of monoclonal antibodies (MAb) against various integrin subunits, and the basement membrane (BM) components laminin and collagen type IV. Selected cases also were immunostained using the avidin-biotin-complex method. The alpha 3 and beta 1 integrin subunits were detected in tumor cells of all the carcinomas. All G1 carcinomas, like normal tubular epithelial cells, expressed the alpha 6 subunit, whereas it was lacking in 20% and 40% of G2 and G3 carcinomas, respectively. Furthermore, when alpha 6 was expressed, a lack of basally polarized organization of the subunit, coupled with disorganization of the BM components, correlated with histologic grade. Another feature that appeared to characterize the more anaplastic tumors was their high level (80%) of the alpha v subunit expression as compared with its absence in the G1 carcinomas. Stromal myofibroblasts, identified by double-labeling with anti-myosin, were often characterized by the expression of the alpha 1, alpha 3, alpha 5 and beta 1 subunits. These results indicate that changes in integrin expression in renal cell carcinomas may be correlated with their degree of histologic malignancy.

Basement Membrane↗
PubMed1991

Human natural killer cells express different integrins and spread on fibronectin.

Human natural killer (NK) cells adhered and most of them also actively spread on cellular fibronectin (cFn), plasma Fn (pFn) and its Mr 120,000-140,000 or Mr 105,000 cell-binding proteolytic Fn-fragments as well as on heparin-binding Fn-fragments containing the alternative cell binding site. The cells did not spread on vitronectin, laminin or collagens. Adhesion on Mr 105,000 Fn fragment containing the cell binding site, could be prevented by the synthetic peptide GRGDS but not by an inactive peptide, whereas adhesion on heparin-binding Fn fragments was unaffected by the peptide. Spreading of the NK cells led to a distinct reorganization of F-actin. Immunoprecipitation with monoclonal antibodies (MoAb) against the beta 1 integrin subunit of radioactively surface-labelled cells revealed a broad polypeptide band of Mr 140,000 under reducing conditions and a polypeptide doublet of Mr 160,000 and Mr 110,000 under non-reducing conditions. Identical polypeptides, corresponding to the alpha- and beta-subunits of the Fn-receptor complex, were bound to the Mr 105,000 chymotryptic Fn-fragment coupled to Sepharose. Similar experiments with small lymphocytes did not reveal any polypeptides. Immunofluorescence results with McAbs suggested that among the alpha-subunits of integrins, the alpha 3, alpha 4, and alpha 5 subunits are expressed in NK cells. The present results suggest that non-activated NK cells, but not small lymphocytes, express beta 1-integrins, and that at least the Fn-receptors alpha 4 beta 1 and alpha 5 beta 1 may function in the adhesion and migration of NK cells.

Cell Adhesion↗
PubMed1991

Distribution of the alpha 1-alpha 6 integrin subunits in human developing and term placenta.

The distribution of the alpha 1-alpha 6 as well as alpha v, beta 1, beta 3 and beta 4 integrin subunits in human first and second trimester and term placentas was studied by indirect immunofluorescence microscopy using a panel of monoclonal antibodies (mAbs). In first and second trimester villi, the alpha 1 and beta 1 integrin subunits were detected in the stromal cells, that were mostly also immunoreactive for desmin. Desmin-positive stromal cells were also found in villi of term placentas, but the stroma was negative for anti-alpha 1 and -beta 1. In the villous trophoblast, anti-alpha 6 and -beta 4 revealed a distinct basal immunoreactivity during all stages of development, whereas immunoreactivity for the alpha 3 and beta 1 subunits emerged during the second and third trimesters. Throughout placental development, endothelia of villous capillaries reacted prominently with anti-alpha 1 and -beta 1. Intermediate trophoblastic cells displayed a somewhat heterogenous immunoreactivity for the beta 1, alpha 1, alpha 3 and alpha 5 integrin subunits, and differed from villous trophoblast also in their lack of expression of the alpha 6 and beta 4 subunits. While nondecidualized endometrial cells displayed weak reactivity for the alpha 1 and beta 1 integrin subunits, the individual decidual cells presented both a basement membrane and a cell surface-confined immunoreactivity for anti-alpha 1, -alpha 3, and -beta 1. The results suggest a role for integrins in placental development, and show that expression of integrins is modulated during the differentiation of trophoblast, villous stroma, and decidual cells. Furthermore, the basal localization of alpha 6 beta 4 and alpha 3 beta 1 integrins suggests that they are employed as basement membrane receptors in the villous trophoblast, and the emergence of the alpha 3 beta 1 complex may reflect that the cytotrophoblast and syncytiotrophoblast recognize the basement membrane differently.

Capillaries↗
PubMed1990

Integrins in human cells and tumors.

We have studied the distribution of the alpha- and beta-subunits of integrins in developing and adult human kidney as well as in selected other tissues and cultured cells. In cultured cells some of the integrin subunits (beta 1, alpha 1, alpha 2 and alpha 5) colocalize with talin at focal adhesions when plated on an appropriate ligand. Similarly, in tissues the polarization of beta 1-integrins in colocalization with talin appears to indicate adhesive complexes, as demonstrated in adult glomeruli. In human kidney, the alpha subunits of integrins were seen to be segment-specifically expressed already in fetal tissues. In glomeruli the integrin alpha 1 subunit characterized mesangial cells while the alpha 2 and alpha 3 subunits showed immunoreactivity in endothelial cells and podocytes, respectively. In renal tubuli, the alpha 6 subunit, complexed with the beta 1 subunit, showed a typical polarized distribution coaligning with the tubular basement membrane while the alpha 3 and alpha 2 subunits were expressed in distal tubular cells. These results suggested that in kidney the alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1 integrins can function as basement membrane receptors. The alpha 5 subunit was nearly lacking in the kidney and it appears to be mainly expressed in some smooth muscle cells. In other tissues distinct patterns in the expression of integrins were found. Thus, in many glandular epithelial cells the alpha 3 beta 1 integrin appeared to function as a basement membrane receptor while in various stratified epithelia and in the breast such a polarized localization could be found for the alpha 6 beta 4 integrin. Finally, although presenting a clearly polarized distribution for beta 1 integrins, none of the alpha subunits could be found in cardiac or skeletal muscle cells and none of the integrins could be revealed in neuronal cells of human developing and adult cerebrum or cerebellum, although neurons in peripheral tissues contained abundantly the alpha 6 beta 1 integrin complex. In human tumors, the tumor cells, including also metastastatic tumors, generally presented the same integrins as their tissues of origin. In some poorly differentiated tumors both a population heterogeneity and even a lack of expression or a disorganization of basement membrane receptor integrins was obvious.

Animals↗