整合素介导小鼠卵内钙离子增加

为了研究整合素是否作为跨膜信号传递受体介导小鼠卵[Ca^2 ]i的变化并探讨其机制。本实验采用甘-精-甘-天冬-丝-脯(GLY-ARG-GLY-ASP-SER-PRO,RGD肽)、纤连蛋A(fibronectin,Fn)及抗整合素α6、β1的单克隆抗体作用于负载了钙探针Fluo-3／AM的去透明带小鼠卵,用激光共聚焦显微镜检测小鼠卵的荧光强度以反映卵[Ca^2 ];用无钙液替代有钙液、或用酪氨酸激酶抑制剂或蛋白激酶C的抑制剂预先作用于卵,然后再观察RGD肽所致卵[Ca^2 ]i的变化。结果显示整合素配体RGD肽或Fn作用于去透明带小鼠卵可引起卵[Ca^2 ]i增加,增加的程度与精子作用相似;去除培养液中的Ca^2 后,再用RGD肽、Fn作用仍可引起卵[Ca^2 ]i增加：用功能性的抗小鼠整合素α6、β1的单克隆抗体也可引起不同程度的卵[Ca^2]i增加,尤其以抗小鼠整合素α6、β1单克隆抗体的作用明显;用酪氨酸激酶抑制剂预先作用于鼠卵,RGD肽或精子作用都不再引起卵[Ca^2 ]i增加;蛋白激酶C抑制剂预先作用鼠卵,RGD肽及Fn也不再引起卵[Ca^2 ]i增加。实验证明。小鼠卵膜整合素与其配体结合可使卵内贮存钙离子释放,引起卵[Ca^2 ]i增加这一卵激活的早期事件;整合素介导小鼠卵激活需要酪氨酸激酶信号转导途径的参与;蛋白激酶C也参与了整合素介导的卵激活。     

骨桥蛋白与生殖

骨桥蛋白是细胞外基质的重要成分,它含有RGD序列,其主要受体为整合素αVβ3,二者相互作用共同参与介导细胞的聚集、黏附、增殖和迁移及免疫调节一系列重要过程.研究发现子宫内膜腺上皮细胞表达骨桥蛋白和其受体整合素αVβ3,且其在子宫内膜种植窗口期分泌至宫腔,推测与内膜容受性的建立及受精卵的种植有关.     

表面等离激元共振生物传感器研究整合素蛋白αⅡbβ3与RGD多肽的特异结合

整合素蛋白αⅡbβ3是血小板上的一种钙依赖性膜受体,其胞外结构域可与含有RGD序列的肽链特异结合.通过将含有NTA-DOGS的磷脂单分子层膜转移到50 nm厚的金膜上,制备了一种含有NTA头部的表面等离激元共振(SPR)传感器敏感膜.设计并合成了含有6个组氨酸和RGD基团的His-tagged短肽P1,并利用SPR生物传感器,对整合素蛋白与含有RGD配基的支撑平面膜的特异相互作用以及Ca2+、Mn2+对该相互作用的影响进行了研究.结果表明,NTA敏感膜能很好地将P1锚定在支撑平面膜表面,并能够保证P1维持一个有效的定向.将Ca2+从低结合位点去除或加入Mn2+都能够增加整合素蛋白的配基结合活性.二价阳离子对整合素蛋白配基结合能力的调节作用,可能在整合素发挥其生理功能的过程中具有重要的意义.     

G蛋白与整合素αⅡbβ3的双向信号转导

整合素是一类细胞表面受体家族分子,通过双向信号转导参与细胞与细胞外基质、细胞与细胞的粘附以及细胞的迁移.整合素αⅡbβ3(GPⅡb-Ⅲa)特异表达于巨核/血小板系,并且是其含量最多的膜糖蛋白,介导血小板的粘附、伸展、聚集等.G蛋白在整合素αⅡbβ3双向信号转导中发挥重要作用,其中较受关注的是:异源三聚体G蛋白和小G蛋白Rap1参与整合素αⅡbβ3的内向外信号转导;小G蛋白(Rho A、Rac等)和Gα13参与整合素αⅡbβ3的外向内信号转导.在蛋白质结构与功能关系的层面,本文总结了G蛋白的结构、分类、功能以及近年来G蛋白在整合素αⅡbβ3双向信号转导中作用的研究进展.     

双靶点分子探针68Ga-RGD-BBN用于乳腺癌的microPET显像

精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)和蛙皮素(bombesin,BBN)多肽分别能够特异性识别整合素αvβ3和胃泌素释放肽受体(gastrin releasing peptide receptor,GRPR).我们用谷氨酸将RGD和BBN连接制备成RGD-BBN异源二聚体多肽,用双功能连接剂NOT...     

表面等离激元共振生物传感器研究整合素蛋白αⅡbβ3与RGD多肽的特异结合

整合素蛋白α_Ⅱbβ₃是血小板上的一种钙依赖性膜受体,其胞外结构域可与含有RGD序列的肽链特异结合.通过将含有NTA-DOGS的磷脂单分子层膜转移到50 nm厚的金膜上,制备了一种含有NTA头部的表面等离激元共振(SPR)传感器敏感膜.设计并合成了含有6个组氨酸和RGD基团的His-tagged短肽P1,并利用SPR生物传感器,对整合素蛋白与含有RGD配基的支撑平面膜的特异相互作用以及Ca²⁺、Mn²⁺对该相互作用的影响进行了研究.结果表明,NTA敏感膜能很好地将P1锚定在支撑平面膜表面,并能够保证P1维持一个有效的定向.将Ca²⁺从低结合位点去除或加入Mn²⁺都能够增加整合素蛋白的配基结合活性.二价阳离子对整合素蛋白配基结合能力的调节作用,可能在整合素发挥其生理功能的过程中具有重要的意义.     

整合素介导小鼠卵内骨架蛋白的重组

研究整合素与卵内骨架蛋白的联系及其是否介导骨架蛋白的重组进而参与卵的激活过程. 5 ~ 8周龄雌性昆明鼠行超数排卵, 用透明质酸酶及MEM酸性液分别去除卵丘细胞及透明带. 用整合素配体的活性片段RGD肽(GLY-ARG-GLY-ASP-SER-PRO, 0.5 mg/mL)与小鼠卵在体外共培养, 以RGE肽(GLY-ARG-GLY-SER-PRO, 0.5 mg/mL)和获能小鼠精子作为对照. 用抗骨架蛋白actin抗体对实验及对照卵行免疫染色, 共聚焦显微镜观察卵内actin的变化, 并对实验及对照卵进行比较. 结果为: (ⅰ) 新鲜排出及经体外培养两小时的两个对照组小鼠卵的免疫染色呈阴性或卵周仅有很弱的荧光带; (ⅱ) RGE肽作用的小鼠卵免疫染色的征象与空白对照没有明显差别; (ⅲ) RGD肽作用不同时间组卵的荧光强度明显增强, 尤以卵周荧光带强而厚, 有的卵周荧光呈不对称分布, 此征象与精子作用后的免疫染色结果相似. 结果表明, 卵膜整合素与卵内骨架蛋白有着直接联系, 整合素配体与卵膜整合素受体结合可能介导卵内骨架蛋白的重组, 从而参与受精过程中的跨膜信号转导过程, 并诱导某些卵激活事件的发生.     

CXCL12过表达促进骨髓基质干细胞整合素αv／β3介导的黏附和增殖

研究利用骨髓基质干细胞移植治疗急性心肌梗死时趋化因子CXCL12过表达对由整合素介导αv/β3的干细胞黏附和增殖过程的影响.采用重组DNA技术使得骨髓基质干细胞过表达趋化因子CXCL12,采用western blot法检测CXCL12过表达后骨髓基质干细胞整合素αv/β3表达量的变化.在体外通过黏附实验观察趋化因子CXCL12过表达对整合素介导的细胞与细胞外基质黏附过程的影响,并在心肌梗死大鼠模型中通过检测报告基因观测CXCL12对移植后整合素介导骨髓基质干细胞增殖的作用.基因重组后骨髓基质干细胞过表达了具有生物活性的趋化因子CXC12,趋化因子CXCL12过表达使骨髓基质干细胞整合素αv/β3表达明显增多,并促进了整合素介导的细胞与细胞外基质黏附.CXCL12还使细胞移植后位于梗死区的细胞数量增多.且这一作用也与整合素αv/β3有关.CXCL12过表达通过促进骨髓基质干细胞整合素αv/β3表达提高了移植干细胞黏附和增殖能力,有利于骨髓基质干细胞移植后在心肌梗死区域的生长和分化.     

人整合素β3亚基真核表达载体的构建及表达

构建人整合素β3亚基全读码框(ORF)基因真核表达载体,为探讨整合素β3作为汉坦病毒(Hantavirus,HV)受体的特异性奠定基础.根据已公布的序列设计引物,用PCR方法从原核质粒中扩增出人整合素分子β3亚基ORF基因,应用TA克隆将其插入pcDNA3.1/V5-His-TOPO载体中,采用酶切和PCR鉴定,选取初筛正向插入的阳性克隆进行测序,序列分析表明与公布的人整合素β3亚基ORF核苷酸序列基本一致,并通过脂质体介导转染至CHO细胞中进行瞬时表达,经间接免疫荧光法检测证实pcDNA3.1-β3能在宿主细胞中高效表达.     

可溶性纤维连接蛋白可活化RhoA并引起癌细胞骨架相关改变

细胞外基质中的纤维连接蛋白可以使细胞表面的整合素受体聚集起来,引起RhoA介导的信号通路的活化,从而导致细胞骨架的重组和细胞迁移的调节。然而,大部分纤维连接蛋白以可溶形式存在于血浆中,这些可溶性纤维连接蛋白是否有相似的效应仍有待于进一步的研究。实验发现,向细胞培养液中加入可溶性纤维连接蛋白,可使胃癌细胞系SGC-7901中的RhoA由GDP结合的非活性形式转变为GTP结合的活性形式,与其底物结合的量增加,而α5β1整合素的抗体可以阻断这一活化过程;可溶性纤维连接蛋白可诱导细胞聚合体形式的肌动蛋白(F-肌动蛋白)的形成。在人前列腺癌细胞系PC-3中,可溶性纤维连接蛋白可引起细胞从多角形向圆形的形态改变,α5β1整合素的抗体可阻断这一改变。以上结果显示可溶性纤维连接蛋白能与α5β1整合素结合并诱导RhoA介导的信号转导。     

Inhibition of cell adhesion by high molecular weight kininogen

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.     

Selective regulation of integrin--cytoskeleton interactions by the tyrosine kinase Src

Cell motility on extracellular-matrix (ECM) substrates depends on the regulated generation of force against the substrate through adhesion receptors known as integrins. Here we show that integrin-mediated traction forces can be selectively modulated by the tyrosine kinase Src. In Src-deficient fibroblasts, cell spreading on the ECM component vitronectin is inhibited, while the strengthening of linkages between integrin vitronectin receptors and the force-generating cytoskeleton in response to substrate rigidity is dramatically increased. In contrast, Src deficiency has no detectable effects on fibronectin-receptor function. Finally, truncated Src (lacking the kinase domain) co-localizes to focal-adhesion sites with alpha v but not with beta 1 integrins. These data are consistent with a selective, functional interaction between Src and the vitronectin receptor that acts at the integrin-cytoskeleton interface to regulate cell spreading and migration.     

A recombinant, arginine-glycine-aspartic acid (RGD) motif from foot-and-mouth disease virus binds mammalian cells through vitronectin and, to a lower extent, fibronectin receptors

The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (_Vβ₃/β₅) and fibronectin (₅β₁) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.     

Inhibition of fibrinogen binding to GP IIb-IIIa by a GP IIIa peptide.

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) mediates platelet aggregation and is a member of the cytoadhesin family of receptors that bind adhesive proteins such as fibrinogen, fibronectin, and von Willebrand factor. Despite the wide range of cell-substrate interactions mediated by these receptors, ligand binding domains have not yet been identified on any of the integrins. The present study was designed to determine potential fibrinogen binding domain(s) on the GP IIb-IIIa complex. Synthetic peptides derived from residues 1-288 of the amino-terminal portion of GP IIIa were tested for their abilities to block the binding of fibrinogen to purified GP IIb-IIIa in a solid-phase microtiter assay. Two overlapping peptides encompassing residues 204-229 of GP IIIa were identified which blocked fibrinogen binding in this assay. Polyclonal antibodies to these peptides blocked fibrinogen binding to purified GP IIb-IIIa as well as platelet aggregation. The overlapping residues of these two peptides GP IIIa (211-222), SVSRNRDAPEGG-NH2, blocked the binding of fibronectin, von Willebrand factor, and vitronectin to purified GP IIb-IIIa. Finally, direct binding of GP IIIa (204-229) to fibrinogen and fibronectin was demonstrated by enzyme-linked immunosorbent assay. We conclude from these studies that the amino acid sequence 211-222 of GP IIIa is critically involved in adhesive protein binding, and may represent an important portion of the GP IIb-IIIa ligand binding domain.     

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

Heparin II domain of fibronectin uses alpha4beta1 integrin to control focal adhesion and stress fiber formation, independent of syndecan-4

Co-signaling events between integrins and cell surface proteoglycans play a critical role in the organization of the cytoskeleton and adhesion forces of cells. These processes, which appear to be responsible for maintaining intraocular pressure in the human eye, involve a novel cooperative co-signaling pathway between alpha5beta1 and alpha4beta1 integrins and are independent of heparan sulfate proteoglycans. Human trabecular meshwork cells isolated from the eye were plated on type III 7-10 repeats of fibronectin (alpha5beta1 ligand) in the absence or presence of the heparin (Hep) II domain of fibronectin. In the absence of the Hep II domain, cells had a bipolar morphology with few focal adhesions and stress fibers. The addition of the Hep II domain increased cell spreading and the numbers of focal adhesions and stress fibers. Cell spreading and stress fiber formation were not mediated by heparan sulfate proteoglycans because treatment with chlorate, heparinase, or soluble heparin did not prevent Hep II domain-mediated cell spreading. Cell spreading and stress fiber formation were mediated by alpha4beta1 integrin because soluble anti-alpha4 integrin antibodies inhibited Hep II domain-mediated cell spreading and soluble vascular cell adhesion molecule-1 (alpha4beta1 ligand)-induced cell spreading. This is the first demonstration of the Hep II domain mediating cell spreading and stress fiber formation through alpha4beta1 integrin. This novel pathway demonstrates a cooperative, rather than antagonistic, role between alpha5beta1 and alpha4beta1 integrins and suggests that interactions between the Hep II domain and alpha4beta1 integrin could modulate the strength of cytoskeleton-mediated processes in the trabecular meshwork of the human eye.     

Integrin beta 1- and beta 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms

Human umbilical vein endothelial cell attachment, spreading and migration on collagen and vitronectin are mediated by integrins alpha 2 beta 1 and alpha v beta 3, respectively, and these events take place in the absence of cytokines, growth factors, or chemoattractants. Cell attachment and spreading on these ligands occur in the absence of extracellular calcium, as does migration on collagen. In contrast, vitronectin-mediated migration is absolutely dependent on the presence of extracellular calcium. Cell contact with immobilized vitronectin or anti-alpha v beta 3 mAbs promotes a measurable rise in [Ca2+]i which requires an extracellular calcium source, whereas collagen, or anti- alpha 2 beta 1 mAbs fail to promote this signaling event. In fact, vitronectin-mediated migration and the rise in intracellular calcium showed the same dose dependence on extracellular calcium. While vitronectin and collagen differ in their ability to induce a calcium influx both ligands or antibodies to their respective integrins promote an equivalent increase in intracellular pH consistent with activation of the Na/H antiporter an event independent of extracellular calcium. These results support two salient conclusions. Firstly, collagen and vitronectin, through their respective integrins, promote distinct intracellular signaling events. Secondly, the alpha v beta 3 specific influx of calcium is not required for cell spreading yet appears to facilitate cellular migration on vitronectin.     

A novel antithrombotic role for high molecular weight kininogen as inhibitor of plasminogen activator inhibitor-1 function

The adhesive glycoprotein vitronectin (VN) forms a function-stabilizing complex with plasminogen activator inhibitor-1 (PAI-1), the major fibrinolysis inhibitor in both plasma and vessel wall connective tissue. VN also interacts with two-chain high molecular weight kininogen (HKa), particularly its His-Gly-Lys-rich domain 5, and both HKa and PAI-1 are antiadhesive factors that have been shown to compete for binding to VN. In this study the influence of HKa and domain 5 on the antifibrinolytic function of PAI-1 was investigated. In a purified system, HKa and particularly domain 5 inhibited the binding of PAI-1 to VN and promoted PAI-1 displacement from both isolated VN as well as subendothelial extracellular matrix-associated VN. The sequence Gly(486)-Lys(502) of HKa domain 5 was identified as responsible for this inhibition. Although having no direct effect on PAI-1 activity itself, HKa domain 5 or the peptide Gly(486)-Lys(502) markedly destabilized the VN.PAI-1 complex interaction, resulting in a significant reduction of PAI-1 inhibitory function on plasminogen activators, resembling the effect of VN antibodies that prevent stabilization of PAI-1. Furthermore, high affinity fibrin binding of PAI-1 in the presence of VN as well as the VN-dependent fibrin clot stabilization by the inhibitor were abrogated in the presence of the kininogen forms mentioned. Taken together, our data indicate that the peptide Gly(486)-Lys(502) derived from domain 5 of HKa serves to interfere with PAI-1 function. Based on these observations potential low molecular weight PAI-1 inhibitors could be designed for the use in therapeutic interventions against thromboembolic complications.     

Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function

Adhesion and signaling by integrins require their dynamic association with nonintegrin membrane proteins. One such protein, the glycolipid-anchored urokinase receptor (uPAR), associates with and modifies the function of the beta(2)-integrin Mac-1 (CD11b/CD18). In this study, a critical non-I-domain binding site for uPAR on CD11b (M25; residues 424-440) is identified by homology with a phage display peptide known to bind uPAR. Recombinant soluble uPAR and cells expressing uPAR bound to immobilized M25, binding being promoted by urokinase and blocked by soluble M25, but not a scrambled control or homologous peptides from other beta(2)-associated alpha-chains. Mac-1, but not a mutated Mac-1 in which M25 was replaced with the homologous sequence of CD11c, co-precipitated with uPAR. In the beta-propeller model of alpha-chain folding, M25 spans an exposed loop on the ligand-binding, upper surface of alphaM, identifying uPAR as an atypical alphaM ligand. Although not blocking ligand binding to Mac-1, M25 (25-100 microM) inhibited leukocyte adhesion to fibrinogen, vitronectin, and cytokine-stimulated endothelial cells. M25 also blocked the association of uPAR with beta(1)-integrins and impaired beta(1)-integrin-dependent spreading and migration of human vascular smooth muscle cells on fibronectin and collagen. These observations indicate that uPAR associates with integrins directly and that disruption of this association broadly impairs integrin function, suggesting a novel strategy for regulation of integrins in the settings of inflammation and tumor progression.