RESUMEN
A recombinant integrin expression system has been created for the large-scale production of αVß5 integrin extracellular domains that take advantage of Fos and Jun dimerization for expression in bacterial, insect, and mammalian cells. This utilizes an all-in-one vector, pQE-TriSystem, with molecular machinery for parallel expression without the need of additional subcloning. Optimal expression in HEK293 cells was determined by a time course analysis. The heterodimer was purified in a one-step nickel column purification scheme, and the sequence and functional state were confirmed by mass spectrometry and inhibition assays, respectively. The yields of αVß5 integrin obtained are in quantities suitable for multiple applications including structural biology and functional assays.
Asunto(s)
Receptores de Vitronectina/genética , Receptores de Vitronectina/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Expresión Génica , Vectores Genéticos/genética , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Receptores de Vitronectina/química , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha(V)beta(3)/beta(5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that alpha(V) integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y(2)R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca(2+). Furthermore, an anti-alpha(V) integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibitor of G(i/o) proteins, partially inhibited Ca(2+) mobilization mediated by the wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Since CD47 has been shown to associate directly with G(i/o) family proteins, these results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Oligopéptidos/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores de Vitronectina/metabolismo , Secuencia de Aminoácidos , Antígenos CD/metabolismo , Antígeno CD47 , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Humanos , Integrinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Unión Proteica , Proteínas Tirosina Quinasas , Receptores Purinérgicos P2/aislamiento & purificación , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y2 , Receptores de Vitronectina/genética , Receptores de Vitronectina/aislamiento & purificación , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
BACKGROUND: Although enamel matrix-derived protein (EMD) can stimulate attachment of human periodontal ligament (HPDL) cells to the root surface, the biological mechanism of this phenomenon is unclear. The purpose of this study was to determine which molecules in EMD are involved in the attachment of HPDL cells, and which types of integrins on the cell surface mediate the interaction between the cells and EMD. METHODS: HPDL explants were obtained from tooth surfaces extracted from 4 individuals, and cells taken from the individual explants were separately harvested and subcultured through as many as 5 passages. Cells were incubated on EMD-coated culture plates with and without neutral antibodies for integrins or RGD-sequence blocking peptides and stained with toluidine blue. Proteins in EMD that were able to induce cell attachment were identified by incubating sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) replicas with HPDL cells; the cell-binding regions were detected by staining the cells with toluidine blue. Characteristics of the cell-binding proteins in the EMD were identified by Western blot analysis. RESULTS: It was shown that anti-alpha(v)beta3 antibody and GRGDSP peptide markedly reduced attachment of HPDL cells to EMD. When the cells were incubated with SDS-PAGE replicas, distinct cell attachment was observed at a molecular mass of approximately 55 kDa. The cell-binding ability of this protein was completely blocked by treatment with anti-alpha(v)beta3 antibody or GRGDSP peptide. In the Western blot analysis, the 55 kDa protein was recognized by anti-bone sialoprotein (BSP) antibody as a single band. CONCLUSIONS: Our study provides the first evidence that the attachment of HPDL cells to EMD can be mediated by interaction between a BSP-like molecule and integrin alpha(v)beta3 on the cell surface.
Asunto(s)
Proteínas del Esmalte Dental/fisiología , Ligamento Periodontal/citología , Receptores de Vitronectina/fisiología , Sialoglicoproteínas/fisiología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Western Blotting , Adhesión Celular , Técnicas de Cultivo de Célula , Colorantes , Proteínas del Esmalte Dental/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Sialoproteína de Unión a Integrina , Peso Molecular , Oligopéptidos/antagonistas & inhibidores , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Vitronectina/aislamiento & purificación , Sialoglicoproteínas/aislamiento & purificación , Estadística como Asunto , Cloruro de TolonioRESUMEN
We have used recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha.beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.
Asunto(s)
Antígenos CD/metabolismo , Proteínas de Unión al Calcio , Proteínas Portadoras/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Antígenos CD/genética , Sitios de Unión , Plaquetas , Calcio/farmacología , Proteínas Portadoras/genética , Cationes Bivalentes/farmacología , Citoplasma , Dimerización , Fosfatasa 2 de Especificidad Dual , Fibrinógeno/metabolismo , Humanos , Integrina beta3 , Ligandos , Manganeso/farmacología , Mutación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/aislamiento & purificación , Glicoproteínas de Membrana Plaquetaria/genética , Unión Proteica/efectos de los fármacos , Proteína Fosfatasa 2 , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Vitronectina/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Alphavbeta3 is the major receptor mediating the attachment of osteoclasts to bone surface and plays a critical role in bone resorption and remodeling. Interfering with alphavbeta3 binding inhibits osteoclast-mediated bone resorption, and thus demonstrates the potential utility of alphavbeta3 antagonists for therapy of osteoporosis. This report describes the generation of an alphavbeta3 affinity column which was created to enable screening of collections of large numbers of ligands, e.g., combinatorial libraries (previously prepared by us), to sort and identify ligands with the highest affinity for alphavbeta3. We demonstrate that covalent immobilization of the heterodimeric alphavbeta3 receptor can be achieved with retention of characteristic ligand binding properties. Human alphavbeta3 was isolated from human embryonic kidney cells (HEK 293) that stably express high levels of the recombinant receptor and then affinity purified to homogeneity. Purified alphavbeta3 receptor was linked covalently to activated CH-Sepharose 4B beads. Quantification of immobilized functional receptor was determined by Scatchard analysis. The immobilized functional receptor maintains binding properties similar to the membrane-embedded and soluble receptor. The immobilized receptor also can be used to select the highest affinity ligand from among a mixture of peptides which differ in their binding affinity, structure, and hydrophobicity, both when the peptides are loaded in equimolar concentrations in a mixture and when the concentration of the highest affinity ligand is reduced 10-fold. Liquid chromatography-mass spectrometry was utilized to confirm selective ligand binding and to demonstrate that preferential binding was not due to nonspecific hydrophobic interactions with immobilized alphavbeta3 receptor or the affinity column. This approach may be of general use for affinity-based screening of ligands for other integrins and should enable practical screening of combinatorial libraries containing large numbers of potential ligands for the human alphavbeta3 integrin receptor, including linear peptides, cyclic peptides, and peptidomimetics.
Asunto(s)
Cromatografía de Afinidad/métodos , Receptores de Vitronectina/química , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes/química , Unión Competitiva , Línea Celular , Cromatografía Liquida , Humanos , Péptidos y Proteínas de Señalización Intercelular , Riñón/citología , Riñón/embriología , Espectrometría de Masas , Microesferas , Péptidos/metabolismo , Péptidos Cíclicos/metabolismo , Receptores de Vitronectina/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sensibilidad y EspecificidadRESUMEN
Angiogenesis, the formation of new blood vessels, is a requirement for malignant tumor growth and metastasis. In the absence of angiogenesis, local tumor expansion is suppressed at a few millimeters and cells lack routes for distant hematogenous spread. Clinical studies have demonstrated that the degree of angiogenesis is correlated with the malignant potential of several cancers, including breast cancer and malignant melanoma. Moreover, the expression of a specific angiogenesis marker, the endothelial integrin alphaVbeta3, has been shown to correlate with tumor grade. However, studies of tumor angiogenesis such as these have generally relied on invasive procedures, adequate tissue sampling and meticulous estimation of histologic microvessel density. In the present report, we describe a novel approach to detecting angiogenesis in vivo using magnetic resonance imaging (MRI) and a paramagnetic contrast agent targeted to endothelial alphaVbeta3 via the LM609 monoclonal antibody. This approach provided enhanced and detailed imaging of rabbit carcinomas by directly targeting paramagnetic agents to the angiogenic vasculature. In addition, angiogenic 'hot spots' not seen by standard MRI were detected. Our strategy for MR imaging of alphaVbeta3 thus represents a non-invasive means to assess the growth and malignant phenotype of tumors.
Asunto(s)
Carcinoma/irrigación sanguínea , Imagen por Resonancia Magnética/métodos , Neovascularización Patológica/diagnóstico , Receptores de Vitronectina/aislamiento & purificación , Animales , Anticuerpos Monoclonales , Medios de Contraste , Sondas Moleculares , ConejosRESUMEN
Integrins and growth factor receptors act synergistically to modulate cellular functions. The alphavbeta3 integrin and the platelet-derived growth factor receptor have both been shown to play a positive role in cell migration. We show here that a platelet derived growth factor-BB gradient stimulated migration of rat microvascular endothelial cells on vitronectin (9.2-fold increase compared to resting cells) in a alphavbeta3 and RGD-dependent manner. In contrast, this response was not observed on a beta1 integrin ligand, laminin; background levels of migration, in response to a platelet derived growth factor-BB gradient, were observed on this substrate or on bovine serum albumin (2.4- or 2.0-fold, respectively). Comparable results were obtained using NIH-3T3 cells. Platelet derived growth factor-BB did not change the cells' ability to adhere to vitronectin, nor did it stimulate a further increase in proliferation on vitronectin versus laminin. In addition, platelet derived growth factor-BB stimulation of NIH-3T3 cells did not alter the ability of alphavbeta3 to bind RGD immobilized on Sepharose. The alphavbeta3 integrin and the platelet derived growth factor receptor-beta associate in both microvascular endothelial cells and NIH-3T3 cells, since they coprecipitated using two different antibodies to either alphavbeta3 or to the platelet derived growth factor receptor-beta. In contrast, beta1 integrins did not coprecipitate with the platelet derived growth factor receptor-beta. These results point to a novel pathway, mediated by the synergistic activity of alphavbeta3 and the platelet derived growth factor receptor-beta, that regulates cell migration and, therefore, might play a role during neovessel formation and tissue infiltration.
Asunto(s)
Movimiento Celular/fisiología , Receptores del Factor de Crecimiento Derivado de Plaquetas/fisiología , Receptores de Vitronectina/metabolismo , Células 3T3 , Animales , Antígenos CD/análisis , Becaplermina , Adhesión Celular , División Celular , Células Cultivadas , Endotelio Vascular/citología , Humanos , Integrina alfaV , Laminina , Ratones , Oligopéptidos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Ratas , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Vitronectina/aislamiento & purificación , VitronectinaRESUMEN
Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38-61 and 82-101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-alphavbeta3 antibodies prevent cell adhesion to FGF-2. Also, purified human alphavbeta3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-alphavbeta3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with alphavbeta3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.
Asunto(s)
Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Receptores de Vitronectina/metabolismo , Secuencias de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Calcio/farmacología , Bovinos , Adhesión Celular/efectos de los fármacos , Línea Celular , Cricetinae , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/química , Fibronectinas/metabolismo , Fibronectinas/farmacología , Heparina/farmacología , Humanos , Mitógenos/antagonistas & inhibidores , Mitógenos/química , Mitógenos/metabolismo , Mitógenos/farmacología , Oligopéptidos/farmacología , Plásticos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/inmunología , Receptores de Vitronectina/aislamiento & purificación , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Eliminación de Secuencia/genética , Solubilidad , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Vitronectina/metabolismo , Vitronectina/farmacologíaRESUMEN
Osteoclastic bone degradation requires intimacy between the matrix and the resorptive cell. While the precise role the integrin alpha(v)beta3 plays in the process is not yet understood, occupancy of the heterodimer by soluble ligand or by blocking antibody effectively inhibits bone resorption in vitro and in vivo, suggesting that alpha(v)beta3 blockade may prevent postmenopausal osteoporosis. Thus, we identified a synthetic chemical peptide mimetic, beta-[2-[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-+ ++oxoethyl]amino-3-pyridinepropanoic acid, bistrifluoroacetate (SC56631) based upon the alpha(v)beta3 ligand, Arg-Gly-Asp (RGD), which recognizes the isolated integrin, and its relative, alpha(v)beta5, as effectively as does the natural peptide. The mimetic dampens osteoclastic bone resorption in vitro and in vivo. Most importantly, intravenous administration of the mimetic prevents the 55% loss of trabecular bone sustained by rats within 6 wk of oophorectomy. Histological examination of bones taken from SC56631-treated, oophorectomized animals also demonstrates the compound's bone sparing properties and its capacity to decrease osteoclast number. Thus, an RGD mimetic prevents the rapid bone loss that accompanies estrogen withdrawal.
Asunto(s)
Resorción Ósea/prevención & control , Integrinas/antagonistas & inhibidores , Osteoporosis/prevención & control , Péptidos/síntesis química , Péptidos/farmacología , Aminoácidos/metabolismo , Animales , Huesos/efectos de los fármacos , Calcitonina/farmacología , Calcio/metabolismo , Adhesión Celular , Células Cultivadas , Dentina/metabolismo , Femenino , Humanos , Oligopéptidos/química , Osteoclastos/efectos de los fármacos , Osteoporosis/sangre , Ovariectomía , Péptidos/administración & dosificación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/aislamiento & purificación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Ratas , Receptores de Vitronectina/aislamiento & purificación , Receptores de Vitronectina/metabolismo , Vitronectina/aislamiento & purificación , Vitronectina/metabolismoRESUMEN
The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily that mediates cell attachment on arginine-glycine-aspartic acid (RGD)-containing adhesive proteins. A solid-phase microtiter assay was developed to investigate the binding properties of purified alpha v beta 3, using tritiated [3H]SK&F-107260 as the radiolabeled ligand. alpha v beta 3, purified from human platelets, human placenta, and chicken osteoclasts, bound [3H]SK&F-107260 saturably and specifically. Saturation binding studies using platelet alpha v beta 3 revealed a single class of high affinity binding sites, exhibiting a Kd of 1.44 nM and Bmax of 0.20 mol of [3H]SK&F-107260/mol of alpha v beta 3. [3H]SK&F-107260 binding was inhibited by a variety of RGD-containing peptides and by the snake venom protein echistatin, whereas an RGE-containing peptide and four nonpeptide fibrinogen receptor (alpha IIb beta 3) antagonists failed to do so. This study shows that alpha v beta 3 exhibits distinct ligand specificity from the structurally homologous fibrinogen receptor, alpha IIb beta 3. The relative potencies of the RGD-containing peptides in inhibiting [3H]SK&F-107260 binding to alpha v beta 3 were the same as their relative potencies in inhibiting biotinylated-fibrinogen binding to the receptor. alpha v beta 3 purified from chicken osteoclasts and human placenta bound [3H]SK&F-107260 with similar affinities and displayed the same pharmacological profile as the platelet vitronectin receptor. The alpha v beta 3 antagonists inhibited the attachment of MG63 human osteosarcoma cells or rat osteoclasts to recombinant rat osteopontin. The rank order of potency of the antagonists in the cell adhesion assays was similar to that of the receptor binding assay, suggesting that the purified alpha v beta 3-[3H]SK&F-107260 binding assay is a valid reflection of the ligand binding to alpha v beta 3 on cell systems.
Asunto(s)
Adhesión Celular , Oligopéptidos/farmacología , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Inhibidores de Agregación Plaquetaria/metabolismo , Receptores de Vitronectina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Plaquetas/metabolismo , Pollos , Cromatografía de Afinidad , Femenino , Humanos , Ligandos , Datos de Secuencia Molecular , Oligopéptidos/química , Osteoclastos/metabolismo , Osteopontina , Osteosarcoma , Placenta/metabolismo , Embarazo , Ensayo de Unión Radioligante , Ratas , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Sialoglicoproteínas/metabolismo , TritioRESUMEN
Vitronectin receptor (alphavbeta3 integrin) is present on the surface of many types of cells. We describe a simple, fast, and reliable method of purification of recombinant human alphavbeta3 from Chinese hamster ovary (CHO) cells transfected with alphavbeta3 (VNRC3 cells). The method consists of two steps: lysis of the cells and affinity chromatography of the lysate on a GRGDSPK-Sepharose column. The yield of the procedure was about 79%. The purified receptor migrated as two bands on a silver stained SDS-polyacrylamide gel, corresponding to the alphav and beta3 subunits, and was recognized by monoclonal antibodies directed against alphav and the alphavbeta3 complex, but not by monoclonal antibody specific for the alphaIIbbeta3 complex. This receptor also bound to immobilized vitronectin, von Willebrand factor, and echistatin. However, binding to immobilized fibrinogen was not observed. Purified recombinant alphavbeta3 demonstrated greater immunoreactivity with LM 609, an alphavbeta3 complex-specific monoclonal antibody, than alphavbeta3 purified from placenta. As visualized by SDS-polyacrylamide gel electrophoresis, preparations of placenta-derived alphavbeta3 contained several contaminating proteins that were not present in preparations of recombinant alphavbeta3 purified from the transfected CHO cells.