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PubMed2000

Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone.

Osteoclasts are actively motile on bone surfaces and undergo alternating cycles of migration and resorption. Osteoclast interaction with the extracellular matrix plays a key role in the osteoclast resorptive process and a substantial body of evidence suggests that integrin receptors are important in osteoclast function. These integrin receptors bind to the Arg-Gly-Asp (RGD) sequence found in a variety of extracellular matrix proteins and it is well established that the interaction of osteoclast alpha v beta 3 integrin with the RGD motif within bone matrix proteins is important in osteoclast-mediated bone resorption. In this study, we characterized the effects of two synthetic peptidomimetic antagonists of alpha v beta 3, SC-56631 and SC-65811, on rabbit osteoclast adhesion to purified matrix proteins and bone, and on bone resorption in vitro. SC-56631 and SC-65811 are potent inhibitors of vitronectin binding to purified alpha v beta 3. Both SC-56631 and SC-65811 inhibited osteoclast adhesion to osteopontin- and vitronectin-coated surfaces and time-lapse video microscopy showed that osteoclasts rapidly retract from osteopontin-coated surfaces when exposed to SC-56631 and SC-65811. SC-56631 and SC-65811 blocked osteoclast-mediated bone resorption in a dose-responsive manner. Further analysis showed that SC-65811 and SC-56631 reduced the number of resorption pits produced per osteoclast and the average pit size. SC-65811 was a more potent inhibitor of bone resorption and the combination of reduced pit number and size led to a 90% inhibition of bone resorption. Surprisingly, however, osteoclasts treated with SC-65811, SC-56631 or the disintegrin echistatin, at concentrations that inhibit bone resorption did not inhibit osteoclast adhesion to bone. These results suggest that alphavbeta3 antagonists inhibited bone resorption by decreasing osteoclast bone resorptive activity or efficiency but not by inhibiting osteoclast adhesion to bone per se.

Aniline Compounds↗

PubMed2000

Contortrostatin activates ERK2 and tyrosine phosphorylation events via distinct pathways.

We report that cells adhering to contortrostatin show transient increases in activation of Extracellular signal Regulated Kinase 2 (ERK2). The kinetics and degree of activation are similar to cells adhering to fibronectin or vitronectin. We have recently shown that contortrostatin induces tyrosine phosphorylation in tumor cells. Contortrostatin is shown here to stimulate activation of ERK2 in suspended cells, but this activation follows a different dose-response pattern than contortrostatin-induced tyrosine phosphorylation. Since contortrostatin induces tyrosine phosphorylation via alphavbeta3, we explored the effects of an alphavbeta3-blocking antibody, 7E3, on contortrostatin-stimulated ERK2 activation. While 7E3 completely blocks the effect of contortrostatin on tyrosine phosphorylation, this antibody had no effect on activation of ERK2. In cells lacking expression of alphavbeta3, tyrosine phosphorylation was unaffected by contortrostatin treatment, but ERK2 was activated. This is strong evidence that contortrostatin is regulating tyrosine phosphorylation events and ERK2 activation via separate pathways and through different integrin receptors.

Cell Adhesion↗

PubMed2001

Evaluation of endothelial cell integrins after in \ vitro contact with polyethylene terephthalate.

The aim of this research was to evaluate the effect of polyethylene terephthalate (Woven Dacron) on the expression of endothelial integrins. Human umbilical vein endothelial cells were cultured on the material for 24 h. The integrins VLA-2 (alpha2beta1-CD49b/CD29), receptor for laminin and collagen, VLA-5 (alpha5beta1-CD49e/CD29), receptor for fibronectin, VLA-6 (alpha6beta1-CD49f/CD29), receptor for laminin, and alphaVbeta3-CD51/CD61 (receptor for vitronectin) were evaluated by flow cytometry. After contact with polyethylene terephthalate, a slight but significant decrease in the percentage of both CD29 and CD49e positive cells was observed, which suggests a lower number of cells expressing the fibronectin receptor alpha5beta1. Moreover, a significant increase in the mean channel for CD49b and for the vitronectin receptor CD51/CD61 was observed. The reduction in the fibronectin receptor could account for the poor endothelialization observed in vivo on polyethylene terephthalate. The increased expression of the vitronectin receptor, favoring the migration of smooth muscle cells, could give some information about the pathogenesis of intimal hyperplasia, which is a complication of vascular grafts.

Journal Article↗

PubMed2001

Anti-alphavbeta3 antibodies in chronic immune thrombocytopenic purpura.

In chronic immune thrombocytopenic purpura (ITP), anti-GPIIb-IIIa (alphaIIbbeta3) autoantibodies have been detected in serum and/or platelet-associated IgG (PAIgG) and considered as one of the major causes. We examined whether anti-alphavbeta3 antibodies might be present in ITP cases because of the similarity between alphavbeta3 and GPIIb-IIIa (alphaIIbbeta3). Modified antigen capture ELISA (MACE) using human umbilical vein endothelial cells (HUVEC) showed the presence of serum anti-alphavbeta3 antibodies in 23 of 80 ITP patients (29%). Cross-adsorption studies between platelets and HUVEC demonstrated that most of anti-alphavbeta3 and anti-GPIIb-IIIa antibodies exclusively reacted with alphavbeta3 and GPIIb-IIIa, respectively. Platelet-associated anti-GPIIb-IIIa antibodies did not react with alphavbeta3, either. Interestingly, patients having anti-alphavbeta3 antibodies showed significantly lower platelet counts than negative patients. These results indicate the serum anti-alphavbeta3 antibodies are different ones from the classical anti-GPIIb-IIIa (alphaIIbbeta3) antibodies and would provide a new insight into the pathophysiology of ITP as well as the autoantigenic epitopes on beta3 integrins.

Autoantibodies↗

PubMed2004

Fibronectin promotes VEGF-induced CD34 cell differentiation into endothelial cells.

BACKGROUND: Adult endothelial progenitor cells (EPC) may be a useful source for engineering the endothelialization of vascular grafts. However, the optimal factors that promote differentiation of EPCs into endothelium remain to be elucidated. The goal of this current report was to determine which extracellular matrix (ECM) protein might modulate or enhance the effects of EPCs on differentiation into mature endothelium. METHODS: Human EPCs (CD34(+) cells) were cultured in ECM-coated six-well plates in MCDB-131 medium containing vascular endothelial growth factor (VEGF), insulin-like growth factor-1, and basic fibroblast growth factor. After 21 days, differentiated endothelial colonies were confirmed by immunofluorescence for von Willebrand factor (vWF) and vascular-endothelial (VE)-cadherin and mRNA expression of the endothelial markers Flk-1, vWF, and VE-cadherin. Cell migration toward the VEGF-matrix protein combinations was also measured. RESULTS: As judged by positive staining for endothelial markers vWF and VE-cadherin, the combination of VEGF with fibronectin (FN) produced significantly more endothelial colonies (P <.05) than did collagens I or IV or vitronectin. Defined fragments of FN did not enhance VEGF-mediated effects. Fibrinogen produced intermediate stimulation of differentiation. FN also enhanced VEGF-mediated CD34(+) cell migration. Blockade of alpha5beta1, but not alphavbeta3 or alphavbeta5, inhibited both VEGF-mediated CD34(+) cell differentiation and migration. CONCLUSIONS: VEGF and FN together significantly promote the migration and differentiation of CD34(+) cells. This synergism is specific to FN and the alpha5beta1 integrin. Combinations of VEGF and FN may be useful in promoting differentiation of circulating endothelial progenitors into endothelial cells for tissue engineering. Clinical relevance Treatment of injured or diseased tissues with adult stem cells is a promising approach. In particular, bone marrow derived circulating endothelial progenitors (CEP's) have been shown to differentiate into endothelial cells in vitro and promote tissue revascularization of ischemic limbs and myocardium in vivo. Because of the relative ease of obtaining CEP's and as well as its high proliferative rate, CEP's may have clinical potential for endothelialization of prosthetic vascular grafts and revascularization of injured myocardium. However, there is a need to better understand the molecular pathways involved in the proliferation and differentiation of CEP's to take full advantage of its clinical potential.

Adult↗

PubMed2001

Expression of integrins in human cultured mesothelial cells: the roles in cell-to-extracellular matrix adhesion and inhibition by RGD-containing peptide.

Integrins play key roles in cell-to-cell and cell-to-extracellular matrix (ECM) adhesion. We investigated integrin expression on pleural mesothelial cells (PMCs) and the inhibitory effect of arginine-glycine-asparate (RGD)-containing peptide on the adhesion of PMCs to fibronectin and collagen. Using flow cytometry and immunostaining, PMCs freshly isolated from pleural effusions and one mesothelial cell line were screened for different integrins. Intact pleural tissue was evaluated by immunohistochemistry. The adhesion of Met-5A cells to fibronectin and collagen types I, III and IV was assayed with prior treatment of various concentrations of glycine-arginine-glycine aspartate-serine (GRGDS). On primary PMCs, alpha2, alpha3, alpha5, beta1, beta3 and alphavbeta3 were highly expressed (>70%); alpha1 expression was intermediate (30-70%); and alpha4 and alpha6 expressions were low (< 30%). On Met-SA cells, alpha3, alpha5, alpha6 and beta1 were highly expressed (>70%); alpha1 was intermediate (30-70%); and alpha2, alpha4, beta3 and alphavbeta3 were low (<30%). The patterns of immunostaining on pleural tissues were similar to the results of flow cytometry for primary PMCs except for beta3. There was no statistically different expression in various disease states (transudate vs. exudate, benign vs. malignant). The inhibitory effect of GRGDS peptide on Met-5A cell adhesion to all four matrix proteins was dose-dependent.

Adolescent↗

PubMed2006

Novel function of human ADAM15 disintegrin-like domain and its derivatives in platelet aggregation.

INTRODUCTION: A Disintegrin and Metalloproteinase (ADAM) proteins are a family of multifunctional proteins containing disintegrin and metalloproteinase domains that perform both adhesive and proteolytic functions in cell-cell and cell-matrix interactions. ADAM15 is unique among these proteins in having an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain. This motif is known to interact with the integrin alphaIIbbeta3 on platelets. MATERIALS AND METHODS: We cloned and expressed the human ADAM15 disintegrin-like domain and its derivatives in Pichia pastoris, and purified them by chromatographic fractionation. We then characterized the integrin binding specificities and their antiplatelet activities of the proteins. Antiplatelet function was assessed by inhibition of platelet adhesion and aggregation. RESULTS: The yeast-expressed ADAM15 disintegrin-like domains were able to inhibit the binding of alphaIIbbeta3 as well as alphavbeta3 to its biological ligands in a dose-dependent manner. Remarkably, mutation of the three residues proximal to the RGD tripeptide sequence, RPTRGD sequence to NWKRGD, increased its affinity for alphaIIbbeta3. The NWK mutant had a much greater inhibitory action on human platelet aggregation than the original ADAM15 disintegrin-like domain. CONCLUSIONS: The structural context of the RGD tripeptide sequence in the disintegrin domain determines the specificity and affinity of the protein for its binding partners. The human ADAM15 disintegrin-like domain may provide useful information for developing an antithrombotic agent.

ADAM Proteins↗

PubMed2004

[Progress of studies on VEC apoptosis-inducing proteins in snake venom and its mechanism--review].

Hemorrhagic snake venom specially induces apoptosis of VEC (vascular endothelial cells). Five apoptosis-inducing proteins had been purified and characterized from crude snake venom. Two of these are L-amino acid oxidase (LAO), the others belong to metalloprotease/disintegrin family. LAO catalyzes H2O2 production by oxidizing some plasma membrane proteins of VEC, disintegrins interfere with binding of integrins with their ligands. The expression of p53 and bcl-2 increases during VEC apoptosis induced by snake venom, moreover, the mRNA of bcl-2 is spliced into two fragments. It has been proved that one of adhesion-dependent signal molecules, alphavbeta3, and one of phospholipid signal molecules, PC-PLC (phosphatidylcholine-specific phospholipase C), are involved in above apoptosis-inducing signal transudation pathway. These results throw light on finding out specific component from protein is snake venom. This component is able to induce tumor vascular endothelial cells apoptosis. This review summarized progress of research on hemorrhagic snake venoms.

Amino Acid Oxidoreductases↗

PubMed2003

Disruption of the beta3 663-687 disulfide bridge confers constitutive activity to beta3 integrins.

The platelet fibrinogen receptor, integrin alphaIIbbeta3, is a noncovalent heterodimer of glycoproteins IIb and IIIa. This work was aimed at elucidating the role played by the carboxy-terminal extracellular, trans-membrane, and cytoplasmic regions of the glycoprotein beta3 in the formation of functional complexes with alpha subunits. Progressive carboxy-terminal deletions of beta3 revealed that surface exposure of alphaIIbbeta3 or alphavbeta3 could not occur in the absence of the transmembrane domain of beta3. In contrast, internal deletions 616 to 690 of the carboxy-terminal regions of the beta3 ectodomain led to surface exposure of constitu tive active receptors in CHO cells, as indicated by the enhanced rate of cell adhesion to immobilized ligands and spontaneous binding to soluble fibrinogen or activation-dependent antibody PAC-1. The functional analysis of cysteine mutations within the 616 to 690 region of beta3 or chimeric beta3-beta7 subunits revealed that disruption of the C663-C687 disulfide bridge endows constitutive activity to the alphaIIbbeta3 receptor. It is concluded that the carboxy-terminal tail of the beta3 ectodomain, so-called beta tail domain (betaTD), is not essential for cell surface expression of beta3 receptors. However, a basal, nonactivated, low ligand-affinity state of the beta3 integrins demands a normal conformation of this domain.

Animals↗

PubMed1999

EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner.

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.

Amino Acid Sequence↗

PubMed2005

Cyr61 induces gastric cancer cell motility/invasion via activation of the integrin/nuclear factor-kappaB/cyclooxygenase-2 signaling pathway.

PURPOSE: Cysteine-rich 61 (Cyr61/CCN1) is involved in many different types of tumor development and progression. Nonetheless, the role of Cyr61 in human gastric cancer has not yet been fully characterized. EXPERIMENTAL DESIGN: We addressed the issue by immunohistochemical staining of 81 gastric adenocarcinoma specimens. Liposome-mediated transfection was used to introduce a Cyr61 expression vector into gastric cancer AGS cell lines. Transfectants were tested in invasion assay by a Boyden chamber. Furthermore, a cyclooxygenase-2 (COX-2) reporter assay and gel mobility shift assay were done to investigate the potential signal pathway of Cyr61. RESULTS: Patients with gastric adenocarcinoma whose tumor displayed high expression of Cyr61 correlated well with aggressive lymph node metastasis, more advanced tumor stage, histologic diffuse type, and early recurrence. Stable transfection of Cyr61 into the AGS cell line strongly enhanced its invasive activity. The overexpression of Cyr61 into AGS cells significantly increased the expression of COX-2 mRNA, protein, and enzymatic activity. Gel mobility shift assays further showed that the nuclear factor-kappaB (NF-kappaB) pathway was evidently activated in Cyr61-expressing AGS cells. Function-neutralizing antibody to alphavbeta3 but not alphavbeta5 effectively suppressed Cyr61-mediated NF-kappaB activation, COX-2 gene expression, and cell invasiveness. CONCLUSIONS: Cyr61 may contribute to the malignant progression of gastric cancer by promoting tumor cell motility/invasion through up-regulation of the functional COX-2 via an integrin alphavbeta3/NF-kappaB-dependent pathway.

Adenocarcinoma↗

PubMed2000

Expression of vitronectin and its integrin receptors in the synovial membrane-like interface tissue from aseptic loosening of total hip replacement.

OBJECTIVE: To investigate expression of vitronectin (VN) and its integrin (Int) receptors in synovial membrane-like interface tissue (SMLIT) in aseptic loosening of total hip replacement (THR), and the potential role of VN-Int interaction in production of collagenase-3. METHODS: Avidin-biotin-peroxidase complex (ABC) staining was used to detect distribution of VN and Int alphaV, beta3, and beta5 subunits. Immunofluorescence labeling with FITC and TRITC conjugated IgG was used to localize Int beta3 subunit and matrix metalloproteinase (MMP-13) double positive cells in SMLIT. RESULTS: Intensive VN immunoreactivity was found in the lining-like layers, sublining area, and endothelium of SMLIT. Statistical analysis of the VN staining score revealed a significant difference between SMLIT and control synovial membrane. All 3 Int subunits appeared in the lining-like layers and sublining area. The Int beta3 subunit was also detected in giant cells of SMLIT. Int beta5 subunit staining was relatively weak and rarely found in vascular endothelium. Immunofluorescence labeling showed many double positive cells in the lining-like layer and sublining area of SMLIT. CONCLUSION: Expressions of VN and Int alphaVbeta3 and alphaVbeta5 are increased in SMLIT compared with that in OA synovial membrane. Int alphaVbeta3 engagement with VN might play a potential role in local MMP-13 production in SMLIT.

Aged↗

PubMed2005

Electron tomography reveals diverse conformations of integrin alphaIIbbeta3 in the active state.

We used electron tomography to determine the three-dimensional (3D) structure of integrin alphaIIbbeta3 in the active state. We found that we obtained better density maps when we reconstructed a 3D volume for each individual particle in the tilt series rather than to extract the particle-containing subvolumes from a 3D reconstruction of the entire specimen area. The 3D tomographic reconstructions of 100 particles revealed that activated alphaIIbbeta3 adopts many different conformations. An average of all the individual 3D reconstructions nicely accommodated the crystal structure of the alphaVbeta3 headpiece, confirming the locations assigned to the alpha- and beta-subunit in the density map. The most striking finding of our study is the structural flexibility of the lower leg of the beta-subunit as opposed to the conformational stability of the leg of the alpha-subunit. The good fit of the atomic structure of the betaI domain and the hybrid domain in the active state showed that the hybrid domain swings out, and most particles used for tomography are in the active state. Multivariate statistical analysis and classification applied to the set of 3D reconstructions revealed that more than 90% reconstructions are grouped into the classes that show the active state. Our results demonstrate that electron tomography can be used to classify complexes with a flexible structure such as integrins.

Cell Line, Tumor↗

PubMed2005

Stable interaction between alpha5beta1 integrin and Tie2 tyrosine kinase receptor regulates endothelial cell response to Ang-1.

During angiogenic remodeling, Ang-1, the ligand of Tie2 tyrosine kinase, is involved in vessel sprouting and stabilization through unclear effects on nascent capillaries and mural cells. In our study, we hypothesized that the Ang-1/Tie2 system could cross-talk with integrins, and be influenced by the dynamic interactions between extracellular matrix and endothelial cells (ECs). Here, we show that alpha5beta1 specifically sensitizes and modulates Tie2 receptor activation and signaling, allowing EC survival at low concentrations of Ang-1 and inducing persistent EC motility. Tie2 and alpha5beta1 interact constitutively; alpha5beta1 binding to fibronectin increases this association, whereas Ang-1 stimulation recruits p85 and FAK to this complex. Furthermore, we demonstrate that Ang-1 is able to mediate selectively alpha5beta1 outside-in FAK phosphorylation. Thus, Ang-1 triggers signaling pathways through Tie2 and alpha5beta1 receptors that could cross-talk when Tie2/alpha5beta1 interaction occurs in ECs plated on fibronectin. By using blocking antibodies, we consistently found that alpha5beta1, but not alphavbeta3 activation, is essential to Ang-1-dependent angiogenesis in vivo.

Angiopoietin-1↗

PubMed2004

Adhesion capacity and integrin expression by dendritic-like cells generated from acute myeloid leukemia blasts by calcium ionophore treatment.

OBJECTIVE: Dendritic cells (DC) play a key role in initiation of immune responses. In vitro modified acute myeloid leukemia (AML) blasts acquire certain specific features of DC and are suggested as a potential source of anti-leukemia vaccines. AML-DC have been characterized in terms of costimulatory molecule expression and cytokine production. In contrast, migratory capacity of AML-DC, which is a major attribute of DC required for their in vivo function, remains unknown. Here we present data on adhesion properties and profile of integrin expression of AML-DC. MATERIALS AND METHODS: Blasts from nine patients were used to generate AML-DC by calcium ionophore treatment. Adhesion of AML-DC to the major components of the extracellular matrix and the profile of integrin expression was studied using flow cytometry. RESULTS: Similar to their normal counterparts, calcium ionophore-induced AML-DC acquired the ability to bind to fibronectin and in 4 of 7 studied cases to bind to denatured collagen. Adhesion to native collagen remained unchanged during DC-type differentiation of AML blasts. AML-DC and DC obtained from monocytes of healthy donors expressed CD49d, CD49e, alphavbeta3, and alphavbeta5. However, AML-DC from 3 of 8 patients down-regulated CD49d, which plays an important role in cell-to-cell and cell-to-matrix interactions and normally is coexpressed with CD83. CONCLUSION: The results provide further evidence that AML blasts can be induced to display functional properties characteristic for DC and may prove useful for in vivo delivery and presentation of tumor antigens to the immune system. Abnormal CD49d expression and variability in AML-DC adhesion to denatured collagen indicate that motility of AML-DC from individual patients may vary, and a customized approach is essential for evaluating leukemic cell feasibility for vaccine design.

Adult↗

PubMed2006

A non-RGD-based integrin binding peptide (ATN-161) blocks breast cancer growth and metastasis in vivo.

PURPOSE: Integrins are expressed by numerous tumor types including breast cancer, in which they play a crucial role in tumor growth and metastasis. In this study, we evaluated the ability of ATN-161 (Ac-PHSCN-NH2), a 5-mer capped peptide derived from the synergy region of fibronectin that binds to alpha5beta1 and alphavbeta3 in vitro, to block breast cancer growth and metastasis. EXPERIMENTAL DESIGN: MDA-MB-231 human breast cancer cells were inoculated s.c. in the right flank, or cells transfected with green fluorescent protein (MDA-MB-231-GFP) were inoculated into the left ventricle of female BALB/c nu/nu mice, resulting in the development of skeletal metastasis. Animals were treated with vehicle alone or by i.v. infusion with ATN-161 (0.05-1 mg/kg thrice a week) for 10 weeks. Tumor volume was determined at weekly intervals and tumor metastasis was evaluated by X-ray, microcomputed tomography, and histology. Tumors were harvested for histologic evaluation. RESULT: Treatment with ATN-161 caused a significant dose-dependent decrease in tumor volume and either completely blocked or caused a marked decrease in the incidence and number of skeletal as well as soft tissue metastases. This was confirmed histologically as well as radiographically using X-ray and microcomputed tomography. Treatment with ATN-161 resulted in a significant decrease in the expression of phosphorylated mitogen-activated protein kinase, microvessel density, and cell proliferation in tumors grown in vivo. CONCLUSION: These studies show that ATN-161 can block breast cancer growth and metastasis, and provides a rationale for the clinical development of ATN-161 for the treatment of breast cancer.

Adenocarcinoma↗

PubMed2001

Platelet integrin alphaIIbbeta3-ligand interactions: what can we learn from the structure?

Upon vascular injury, platelets initiate interaction with exposed subendothelial matrices through various receptors such as glycoprotein (GP) Ib/IX/V complex, alpha2beta1 integrin, and GPVI/FcRgamma. Although these interactions cannot sustain stable platelet thrombus formation by themselves, they ultimately lead to the activation of alphaIIbbeta3 integrin (GPIIb-IIIa complex [GPIIb-IIIa]), the most abundant receptor in platelets. The alphaIIbbeta3 integrin plays a central role in primary hemostasis by serving as a receptor for fibrinogen and von Willebrand factor (vWf). It establishes a stable interaction with vWf bound to the extracellular matrices and uses fibrinogen as a bridging molecule in platelet aggregate formation. The alphaIIbbeta3 integrin also plays an important role in the pathogenesis of thrombosis. Over the past decades, a tremendous amount of effort has been made to elucidate the ligand-binding mechanisms of alphaIIbbeta3, in part because of its clinical significance. Most of the studies have relied on biochemical analyses of purified alphaIIbbeta3 or recombinant proteins generated in vitro. With the lack of actual 3-dimensional structure, molecular modeling has provided a useful framework for interpreting such experimental data on structure-function correlation of integrin molecules. However, it has also generated disagreement between different models. The aim of this minireview is to summarize the past efforts as well as the recent accomplishments in elucidating the structure/function of alphaIIbbeta3. Finally, we will try to explain all those experimental data using the recently published crystal structure of the extracellular domains of the alphaVbeta3 heterodimeric complex.

Animals↗

PubMed2005

The intermediate filament protein vimentin binds specifically to a recombinant integrin alpha2/beta1 cytoplasmic tail complex and co-localizes with native alpha2/beta1 in endothelial cell focal adhesions.

Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short alpha and beta cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin alpha2beta1 is a major collagen receptor but to date, only few proteins have been shown to interact with the alpha2 cytoplasmic tail or with the alpha2beta1 complex. In order to identify novel binding partners of a alpha2beta1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-alpha2 and GST-Jun alpha2 bound His-tagged calreticulin while GST-beta1 and GST-Fos beta1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun alpha2/GST-Fos beta1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with alphavbeta3-positive focal contacts. Here, we provide evidence that this interaction also occurs with alpha2beta1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.

Amino Acid Sequence↗