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1.
Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151 总被引:4,自引:0,他引:4
Yauch RL Kazarov AR Desai B Lee RT Hemler ME 《The Journal of biological chemistry》2000,275(13):9230-9238
Previously we established that the alpha(3)beta(1) integrin shows stable, specific, and stoichiometric association with the TM4SF (tetraspannin) protein CD151. Here we used a membrane impermeable cross-linking agent to show a direct association between extracellular domains of alpha(3)beta(1) and CD151. The alpha(3)beta(1)-CD151 association site was then mapped using chimeric alpha(6)/alpha(3) integrins and CD151/NAG2 TM4SF proteins. Complex formation required an extracellular alpha(3) site (amino acids (aa) 570-705) not previously known to be involved in specific integrin contacts with other proteins and a region (aa 186-217) within the large extracellular loop of CD151. Notably, the anti-CD151 monoclonal antibody TS151r binding epitope, previously implicated in alpha(3) integrin association, was mapped to the same region of CD151 (aa 186-217). Finally, we demonstrated that both NH(2)- and COOH-terminal domains of CD151 are located on the inside of the plasma membrane, thus confirming a long suspected model of TM4SF protein topology. 相似文献
2.
Dirk LM Schmidt JJ Cai Y Barnes JC Hanger KM Nayak NR Williams MA Grossman RB Houtz RL Rodgers DW 《The Biochemical journal》2008,415(3):417-427
CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin alpha 3 beta 1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin alpha 3 beta 1 remains poorly understood. In the present study, we show that a panel of anti-human CD151 mAbs (monoclonal antibodies) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin alpha 3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin alpha 3 under both low- and high-stringency detergent conditions; Group-2 mAbs co-precipitated integrin alpha 3 under low-stringency conditions; and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 mAbs in particular exhibited increased reactivity toward integrin alpha 3 beta 1-unbound CD151, indicating that the binding sites for Group-1 mAbs are partly blocked by bound integrin alpha 3 beta 1. Epitope mapping using a series of CD151 mutants with substitutions at amino acid residues that are not conserved between human and mouse CD151 revealed that Gly(176)/Gly(177), Leu(191) and Gln(194) comprise epitopes characteristic of Group-1 mAbs. Replacement of short peptide segments, each containing one of these epitopes, with those of other tetraspanins lacking stable interactions with integrin alpha 3 beta 1 demonstrated that the segment from Cys(185) to Cys(192), including Leu(191), was involved in the stable association of CD151 with integrin alpha 3 beta 1, as was the Gln(194)-containing QRD peptide. Taken together these results indicate that two consecutive segments including two Group-1 epitopes, Leu(191) and Gln(194), comprise an interface between CD151 and integrin alpha 3 beta 1, and, along with the epitope including Gly(176)/Gly(177), are concealed by bound integrin. 相似文献
3.
Liu L He B Liu WM Zhou D Cox JV Zhang XA 《The Journal of biological chemistry》2007,282(43):31631-31642
Regulation of cell migration is an important feature of tetraspanin CD151. Although it is well established that CD151 physically associates with integrins, the mechanism by which CD151 regulates integrin-dependent cell migration is basically unknown. Given the fact that CD151 is localized in both the plasma membrane and intracellular vesicles, we found that CD151 and its associated alpha3beta1, alpha5beta1, and alpha6beta1 integrins undergo endocytosis and accumulate in the same intracellular vesicular compartments. CD151 contains a YRSL sequence, a YXXvarphi type of endocytosis/sorting motif, in its C-terminal cytoplasmic domain. Mutation of this motif markedly attenuated CD151 internalization. The loss of CD151 trafficking completely abrogated CD151-promoted cell migration on extracellular matrices such as laminin and diminished the internalization of its associated integrins, indicating a critical role for integrin trafficking in regulating cell motility. In conclusion, the YXXvarphi motif-mediated internalization of CD151 promotes integrin-dependent cell migration by modulating the endocytosis and/or vesicular trafficking of its associated integrins. 相似文献
4.
Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis 总被引:5,自引:0,他引:5
Berditchevski F Gilbert E Griffiths MR Fitter S Ashman L Jenner SJ 《The Journal of biological chemistry》2001,276(44):41165-41174
Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the alpha3beta1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with alpha3beta1, maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu(149) and Glu(213)). Furthermore, the substitution of 11 amino acids (residues 195-205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151-CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin-tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151-alpha3beta1 interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151-alpha3beta1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the "tetraspanin web" can be controlled at various levels by different regions of the protein. 相似文献
5.
A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5
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Winterwood NE Varzavand A Meland MN Ashman LK Stipp CS 《Molecular biology of the cell》2006,17(6):2707-2721
The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration. 相似文献
6.
Chometon G Zhang ZG Rubinstein E Boucheix C Mauch C Aumailley M 《Experimental cell research》2006,312(7):983-995
Laminin-binding integrins form a complex with CD151, a member of the tetraspanin family suggested to be involved in the regulation of cell migration. In the epidermis, CD151 is localized with alpha3beta1 and alpha6beta4 integrins at cell-cell and cell-matrix contacts, respectively, characteristic structures of non-migrating cells. Taking advantage of a monoclonal antibody against CD151, TS151r, which epitope overlaps with the tetraspanin integrin-binding site, we have investigated the role of CD151 in epithelial cell migration. Under standard culture conditions, the migratory capacity of epithelial HaCaT cells on laminins is low, apparently due to endogenous laminin 5. However, challenging HaCaT cells with TS151r allows a re-arrangement of the actin cytoskeleton, dismantling of cell-cell and beta4 integrin-mediated cell-matrix contacts and cell migration. In vivo, free CD151 is absent in resting epithelial cells of interfollicular epidermis, and all CD151 is bound to integrins in intercellular and cell-matrix contacts. By contrast, free CD151 is present at intercellular contacts in the epithelial sheet lining the deeper region of anagen hair follicles, which is considered to contain migrating cells. Together, these results strongly suggest that dissociation of the CD151-integrin complex permits remodeling of epithelial cell interactions with the extracellular matrix and cell migration. 相似文献
7.
Sterk LM Geuijen CA Oomen LC Calafat J Janssen H Sonnenberg A 《The Journal of cell biology》2000,149(4):969-982
CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface.In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components. 相似文献
8.
The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3β1, which mediates rapid tumor cell migration, and integrin α6β4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3β1 and α6β4 integrins and with other tetraspanins, thereby promoting α3β1 and α6β4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi-mediated silencing of endogenous CD151. The interactions of CD151 with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151-integrin association and CD151-tetraspanin association were both important for α3β1 integrin-dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6β4 integrin-dependent cell attachment on laminin-332. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain, which had been thought to be crucial for CD151-integrin interaction, is not essential for CD151-integrin association or for the ability of CD151 to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact. 相似文献
9.
Berditchevski F Odintsova E Sawada S Gilbert E 《The Journal of biological chemistry》2002,277(40):36991-37000
Transmembrane proteins of the tetraspanin superfamily are assembled in multimeric complexes on the cell surface. Spatial orientation of tetraspanins within these complexes may affect signaling functions of the associated transmembrane receptors (e.g. integrins, receptor-type tyrosine kinases). The structural determinants that control assembly of the tetraspanin complexes are unknown. We have found that various tetraspanins and the alpha(3) integrin subunit are palmitoylated. The stability and molecular composition of the palmitoylated alpha(3)beta(1)-tetraspanin complexes are not affected by adhesion. To assess the significance of palmitoylation in the function of the alpha(3)beta(1)-tetraspanin complexes we mapped the sites of palmitoylation for CD151. Mutation of six cysteines, Cys(11), Cys(15), Cys(79), Cys(80), Cys(242), and Cys(243) was necessary to completely abolish palmitoylation of CD151. The association of the palmitoylation-deficient mutant of CD151 (CD151Cys8) with CD81 and CD63 was markedly decreased, but the interaction of the alpha(3)beta(1)-CD151Cys8 complex with phosphatidylinositol 4-kinase was not affected. Ectopic expression of CD151Cys8 in Rat-1 cells impaired the interactions of the endogenous CD63 and CD81 with the alpha(3)beta(1) integrin. Although the expression of the palmitoylation-deficient CD151 does not change cell spreading on the extracellular matrix, the number of focal adhesions increased. Adhesion-induced phosphorylation of PKB/c-Akt is markedly increased in cells expressing a palmitoylation-deficient mutant, thereby providing direct evidence for the role of the tetraspanin microdomains in regulation of the integrin-dependent phosphatidylinositol 3-kinase signaling pathway. In contrast, activation of FAK and ERK1/2 were not affected by the expression of CD151Cys8. Our results demonstrate that palmitoylation of tetraspanins is critical not only for the organization of the integrin-tetraspanin microdomains but also has a specific role in modulation of adhesion-dependent signaling. 相似文献
10.
Dorfleutner A Hintermann E Tarui T Takada Y Ruf W 《Molecular biology of the cell》2004,15(10):4416-4425
In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is beta1 integrin-dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing alphavbeta3 or certain beta1 integrin heterodimers (alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha9beta1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates alpha3beta1-dependent migration on laminin 5. Expression of TF suppresses alpha3beta1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2-dependent phosphorylation of TF. In both cases, release of alpha3beta1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra- and extracellular interactions and phosphorylation regulates TF's function in cell motility. 相似文献
11.
Thorne RF Marshall JF Shafren DR Gibson PG Hart IR Burns GF 《The Journal of biological chemistry》2000,275(45):35264-35275
Lateral association between different transmembrane glycoproteins can serve to modulate integrin function. Here we characterize a physical association between the integrins alpha(3)beta(1) and alpha(6)beta(1) and CD36 on the surface of melanoma cells and show that ectopic expression of CD36 by CD36-negative MV3 melanoma cells increases their haptotactic migration on extracellular matrix components. The association was demonstrated by co-immunoprecipitation, reimmunoprecipitation, and immunoblotting of surface-labeled cells lysed in Brij 96 detergent. Confocal microscopy illustrated the co-association of alpha(3) and CD36 in cell membrane projections and ruffles. A requirement for the extracellular domain of CD36 in this association was shown by co-immunoprecipitation experiments using surface-labeled MV3 melanoma or COS-7 cells that had been transiently transfected with chimeric constructs between CD36 and intercellular adhesion molecule 1 (ICAM-1) or with a truncation mutant of CD36. CD36 is known to engage in signal transduction and to localize to membrane microdomains or rafts in several cell types. Toward a mechanistic explanation for the functional effects of CD36 expression, we demonstrate that in fractionated Triton X-100 lysates of the MV3 cells stably transfected with CD36, CD36 was greatly enriched with the detergent-insoluble fractions that represent plasma membrane rafts. Significantly, when these fractionated lysates were reprobed for endogenous beta(1) integrin, it was found that a 4-fold increase in the proportion of the mature protein was contained within the detergent-insoluble fractions when extracted from the CD36-transfected cells compared with MV3 cells transfected with vector only. These results suggest that in melanoma cells CD36 expression may induce the sequestration of certain integrins into membrane microdomains and promote cell migration. 相似文献
12.
Chandrasekaran S Guo NH Rodrigues RG Kaiser J Roberts DD 《The Journal of biological chemistry》1999,274(16):11408-11416
Thrombospondin-1 (TSP1) is a matricellular protein that displays both pro- and anti-adhesive activities. Binding to sulfated glycoconjugates mediates most high affinity binding of soluble TSP1 to MDA-MB-435 cells, but attachment and spreading of these cells on immobilized TSP1 is primarily beta1 integrin-dependent. The integrin alpha3beta1 is the major mediator of breast carcinoma cell adhesion and chemotaxis to TSP1. This integrin is partially active in MDA-MB-435 cells but is mostly inactive in MDA-MB-231 and MCF-7 cells, which require beta1 integrin activation to induce spreading on TSP1. Integrin-mediated cell spreading on TSP1 is accompanied by extension of filopodia containing beta1 integrins. TSP1 binding activity of the alpha3beta1 integrin is not stimulated by CD47-binding peptides from TSP1 or by protein kinase C activation, which activate alphavbeta3 integrin function in the same cells. In MDA-MB-231 but not MDA-MB-435 cells, this integrin is activated by pertussis toxin, whereas serum, insulin, insulin-like growth factor-1, and ligation of CD98 increase activity of this integrin in both cell lines. Serum stimulation is accompanied by increased surface expression of CD98, whereas insulin-like growth factor-1 does not increase CD98 expression. Thus, the pro-adhesive activity of TSP1 for breast carcinoma cells is controlled by several signals that regulate activity of the alpha3beta1 integrin. 相似文献
13.
Function of the Tetraspanin CD151–α6β1 Integrin
Complex during Cellular Morphogenesis 总被引:5,自引:0,他引:5
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Xin A. Zhang Alexander R. Kazarov Xiuwei Yang Alexa L. Bontrager Christopher S. Stipp Martin E. Hemler 《Molecular biology of the cell》2002,13(1):1-11
Upon plating on basement membrane Matrigel, NIH3T3 cells formed an anastomosing network of cord-like structures, inhibitable by anti-alpha6beta1 integrin antibodies. For NIH3T3 cells transfected with human CD151 protein, the formation of a cord-like network was also inhibitable by anti-CD151 antibodies. Furthermore, CD151 and alpha6beta1 were physically associated within NIH3T3 cells. On removal of the short 8-amino acid C-terminal CD151 tail (by deletion or exchange), exogenous CD151 exerted a dominant negative effect, as it almost completely suppressed alpha6beta1-dependent cell network formation and NIH3T3 cell spreading on laminin-1 (an alpha6beta1 ligand). Importantly, mutant CD151 retained alpha6beta1 association and did not alter alpha6beta1-mediated cell adhesion to Matrigel. In conclusion, the CD151-alpha6beta1 integrin complex acts as a functional unit that markedly influences cellular morphogenesis, with the CD151 tail being of particular importance in determining the "outside-in" functions of alpha6beta1-integrin that follow ligand engagement. Also, antibodies to alpha6beta1 and CD151 inhibited formation of endothelial cell cord-like networks, thus pointing to possible relevance of CD151-alpha6beta1 complexes during angiogenesis. 相似文献
14.
Yang X Kovalenko OV Tang W Claas C Stipp CS Hemler ME 《The Journal of cell biology》2004,167(6):1231-1240
As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (alpha3, alpha6, and beta4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of beta4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient beta4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core alpha6beta4-CD151 complex formation was unaltered. There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading. Notably, beta4 palmitoylation neither increased localization into "light membrane" fractions of sucrose gradients nor decreased solubility in nonionic detergents-hence it does not promote lipid raft association. Instead, palmitoylation of beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation. 相似文献
15.
A functionally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated beta1 integrin 总被引:1,自引:0,他引:1
Gutierrez-Lopez MD Ovalle S Yanez-Mo M Sanchez-Sanchez N Rubinstein E Olmo N Lizarbe MA Sanchez-Madrid F Cabanas C 《The Journal of biological chemistry》2003,278(1):208-218
Tetraspanins associate on the cell membrane with several transmembrane proteins, including members of the integrin superfamily. The tetraspanin CD9 has been implicated in cell motility, metastasis, and sperm-egg fusion. In this study we characterize the first CD9 conformation-dependent epitope (detected by monoclonal antibody (mAb) PAINS-13) whose expression depends on changes in the activation state of associated beta(1) integrins. MAb PAINS-13 precipitates CD9 under conditions that preserve the association of this tetraspanin with integrins, but not under conditions that disrupt these interactions. Induction of activation of beta(1) integrins by temperature, divalent cation Mn(2+), or mAb TS2/16 correlated with enhanced expression of the PAINS-13 epitope on a variety of cells. Through the use of different K562 myeloid leukemia transfectant cells expressing specific members of the beta(1) integrin subfamily we show that the expression of the PAINS-13 epitope depends on CD9 association with alpha(6)beta(1) integrin. The mAb PAINS-13 reactivity has been mapped to the CD9 region comprising residues 112-154 in the NH(2) half of the large extracellular loop. Also, we show that the CD9 conformation recognized by mAb PAINS-13 is functionally relevant in beta(1) integrin-mediated cellular processes including wound healing migration, tubular morphogenesis, cell adhesion and spreading and in signal transduction involving phosphatidylinositol 3-kinase activation. 相似文献
16.
Kabir-Salmani M Fukuda MN Kanai-Azuma M Ahmed N Shiokawa S Akimoto Y Sakai K Nagamori S Kanai Y Sugihara K Iwashita M 《Molecular endocrinology (Baltimore, Md.)》2008,22(3):707-715
CD98 heavy chain (CD98hc) is expressed highly in developing human placental trophoblast. CD98hc is an amino acid transporter and is thought to function in cell fusion, adhesion, and invasion by interacting with integrins. In invasive extravillous trophoblast, alpha(v)beta(3) integrin is expressed in a temporally and spatially specific manner, which prompted us to investigate the potential role of CD98hc in signal transduction of alpha(v)beta(3) integrin. Immunocytochemistry of extravillous trophoblast derived from human placenta revealed that CD98hc colocalized with alpha(v)beta(3) integrin and with alpha(v)beta(3)-associated cytoplasmic proteins including paxillin, vinculin, and focal adhesion kinase. Coimmunoprecipitation of CD98hc and its mutants revealed that the transmembrane domain of CD98hc is necessary for the association of CD98hc with alpha(v)beta(3) integrin. When CD98hc negative liver cells (FLC4) were stably transfected with CD98hc and the extracellular domain of CD98hc was cross-linked by anti-CD98 antibody, FLC4 cells binding affinity to fibronectin and cell motility increased. The anti-CD98 antibody cross-linking promoted actin stress fiber formation and activation of signal transduction downstream of RhoA GTPase, and elevated the phosphorylation of focal adhesion kinase, paxillin, and protein kinase B. Pretreatment of transfected FLC4 cells with specific inhibitors for alpha(v)beta(3)integrin, phosphatidylinositol 3-kinase, and RhoA diminished these effects caused by anti-CD98 antibody cross-linking. These results suggest that notoriously invasive activity of extravillous trophoblast is mediated by CD98hc, which promotes alpha(v)beta(3) integrin-dependent signals. 相似文献
17.
目的研究整合素β1在CD151促脐静脉内皮细胞迁移增殖中的机制。方法通过构建PAAV-CD151质粒及其pAAV-CD151-AAA194-196突变体(QRD)并转染HUVEC,HUVEC分为正常对照组、绿色荧光组(GFP组)、CD151组,QRD组。SRB法检测细胞的增殖,划痕试验检测细胞的迁移能力,Western blot检测CD151、Akt、P-Akt、PI3K及β1的表达。结果1、CD151组与对照组及GFP组相比具有明显促进细胞增殖的能力,而QRD组则显著抑制细胞的增殖,具有显著的统计学意义(P0.05)。2、CD151组与对照组及GFP组相比具有明显促进细胞迁移的能力,而QRD组则显著抑制细胞的迁移,具有显著的统计学意义(P0.05)。3、CD151组β1、PI3K、P-Akt蛋白表达量明显增高,与对照组、GFP组和QRD组相比具有显著差异(P0.05),QRD组蛋白表达量明显降低具有显著差异(P0.05),而总的Akt四组之间无明显差异(P0.05)。结论CD151具有促进血管形成的作用,通过整合素β1上调PI3K的表达,进一步促进Akt的磷酸化,增加Akt的活力,达到促进内皮细胞的迁移和增殖及管状结构的形成,进而促进血管形成。 相似文献
18.
Tetraspanin CD151 associates with laminin-binding α(3)β(1)/α(6)β(1) integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α(3)β(1)/α(6)β(1) integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β(1) integrin and, thereby, increased complex formation between the β(1) family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β(1) integrin complex-stimulating adhesion events, such as α(3)β(1)/α(6)β(1) integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β(1) integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β(1) integrin, only CD151 exhibited the ability to facilitate complex formation between the β(1) family of integrins and small GTPases and stimulate β(1) integrin-dependent activation of small GTPases. These results suggest that CD151 links α(3)β(1)/α(6)β(1) integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation. 相似文献
19.
Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins 总被引:26,自引:0,他引:26
Translocation of conventional protein kinases C (PKCs) to the plasma membrane leads to their specific association with transmembrane-4 superfamily (TM4SF; tetraspanin) proteins (CD9, CD53, CD81, CD82, and CD151), as demonstrated by reciprocal co-immunoprecipitation and covalent cross-linking experiments. Although formation and maintenance of TM4SF-PKC complexes are not dependent on integrins, TM4SF proteins can act as linker molecules, recruiting PKC into proximity with specific integrins. Previous studies showed that the extracellular large loop of TM4SF proteins determines integrin associations. In contrast, specificity for PKC association probably resides within cytoplasmic tails or the first two transmembrane domains of TM4SF proteins, as seen from studies with chimeric CD9 molecules. Consistent with a TM4SF linker function, only those integrins (alpha(3)beta(1), alpha(6)beta(1), and a chimeric "X3TC5" alpha(3) mutant) that associated strongly with tetraspanins were found in association with PKC. We propose that PKC-TM4SF-integrin structures represent a novel type of signaling complex. The simultaneous binding of TM4SF proteins to the extracellular domains of the integrin alpha(3) subunit and to intracellular PKC helps to explain why the integrin alpha3 extracellular domain is needed for both intracellular PKC recruitment and PKC-dependent phosphorylation of the alpha(3) integrin cytoplasmic tail. 相似文献
20.
Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model
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We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha. 相似文献