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1.
Nat Med ; 4(3): 336-40, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9500609

RESUMO

The development and endocrine function of the ovarian corpus luteum (CL) are dependent on the growth of new capillary vessels. Although several molecules have been implicated as mediators of CL angiogenesis, at present there is no direct evidence for the involvement of any. Here we report the unexpected finding that treatment with truncated soluble Flt-1 receptors, which inhibit vascular endothelial growth factor (VEGF) bioactivity, resulted in virtually complete suppression of CL angiogenesis in a rat model of hormonally induced ovulation. This effect was associated with inhibition of CL development and progesterone release. Failure of maturation of the endometrium was also observed. Areas of ischemic necrosis were demonstrated in the corpora lutea (CLs) of treated animals. However, no effect on the preexisting ovarian vasculature was observed. These findings demonstrate that, in spite of the redundancy of potential mediators, VEGF is essential for CL angiogenesis. Furthermore, they have implications for the control of fertility and the treatment of ovarian disorders characterized by hypervascularity and hyperplasia.


Assuntos
Corpo Lúteo/irrigação sanguínea , Fatores de Crescimento Endotelial/farmacologia , Linfocinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Animais , Corpo Lúteo/anatomia & histologia , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/genética , Feminino , Gonadotropinas/farmacologia , Hibridização in Situ Fluorescente , Linfocinas/antagonistas & inibidores , Linfocinas/genética , Ratos , Ratos Sprague-Dawley , Útero/anatomia & histologia , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
4.
J Biol Chem ; 273(6): 3216-22, 1998 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-9452434

RESUMO

Flt-1 is one of two receptor tyrosine kinases through which the angiogenic factor vascular endothelial growth factor (VEGF) functions. Placenta growth factor (PlGF) is an additional ligand for Flt-1. The second immunoglobulin-like domain in the extracellular domain of Flt-1 has previously been identified as the region containing the critical ligand-binding determinants. We analyzed the contribution of charged residues within the first three domains of Flt-1 to ligand binding by alanine-scanning mutagenesis. Domain 2 residues Arg159, Glu208 and His223-Arg224 (together) affect both VEGF and PlGF binding, while Glu137, Lys171, His223, and Arg224 affect PlGF but not VEGF. Several charged residues, especially Asp187, are important in maintaining the structural integrity of domain 2. In addition, some residues in domain 3 contribute to binding (Asp231) or provide for additional discrimination between ligands (Arg280-Asp283).


Assuntos
Fatores de Crescimento Endotelial/química , Imunoglobulinas/química , Linfocinas/química , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases/química , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Humanos , Imunoglobulinas/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Homologia de Sequência de Aminoácidos , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
5.
EMBO J ; 15(18): 4919-27, 1996 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-8890165

RESUMO

Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt-1 and KDR. Flt-1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt-1 has an alternative ligand, placenta growth factor (PlGF). Both Flt-1 and KDR have seven immunoglobulin (Ig)-like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt-1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig-like domain is the major determinant for VEGF-PlGF interaction and that binding to this domain may initiate a signal transduction cascade.


Assuntos
Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Células 3T3 , Sequência de Aminoácidos , Animais , Replicação do DNA/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Transfecção , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio Vascular
6.
J Biol Chem ; 271(49): 31679-87, 1996 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-8940189

RESUMO

The cloning and expression of two new human cDNAs encoding proteins highly related to the far upstream element-binding protein (FBP) are described. FBP, FBP2, and FBP3 comprise a family of single-strand DNA- binding proteins that possess all of the general features of more conventional transcription factors. The FBPs each bind sequence specifically to only one strand of the far upstream element (FUSE; originally identified upstream of c-myc), and each possesses potent activation domains when fused to the GAL4 DNA-binding domain and assayed by transient transfection. Typical of transcription factors, the proteins are most highly related in their central, DNA-binding domains, but extensive homology is also shared within the tyrosine-rich, carboxyl-terminal activation domains. Comparison with GenBank sequences revealed a fourth FBP family member encoded by Caenorhabditis elegans chromosome III, illustrating the high degree of homology in this evolutionarily ancient and conserved family.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Região Organizadora do Nucléolo , Proteínas de Plantas/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição , Proteínas de Ligação a RNA , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans , Sequência Conservada , DNA Complementar/química , Humanos , Dados de Sequência Molecular , Proteínas de Plantas/química , Transativadores/química
7.
J Biol Chem ; 268(35): 26466-75, 1993 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-8253775

RESUMO

Activated T-cell extracts contain an activity (T-AP1) composed of at least two dissociable protein components which bind to the AP1 consensus sequence in the enhancer of the gibbon ape leukemia virus (GALV)-LTR (GALV-TRE). This activity is inducible by 12-O-tetradecanoyl-phorbol-14-acetate (TPA) even in the presence of protein synthesis inhibitors. Although one component of this complex (CORE) is related immunologically and biochemically to junD, it nevertheless displays significant biochemical properties which distinguish CORE from recombinant junD. The second component of the complex, flowthrough, interacts more efficiently with CORE than with recombinant junD. GALV-TRE enhancer activity is increased within 2 h in vivo with T cells treated with TPA in the presence of protein synthesis inhibitors; this increase in enhancer activity is paralleled by the increased GALV-TRE-mediated transcriptional activity present in extracts of these cells. Purified T-cell junD activates GALV-TRE-driven RNA synthesis in vitro. The rapidity and the protein synthesis-independent nature of TPA-induced T-AP1 activation suggests that this complex is involved in the earliest stages of T-cell activation.


Assuntos
Ativação Linfocitária , Proteínas Proto-Oncogênicas c-jun/metabolismo , Linfócitos T/metabolismo , Animais , Sequência de Bases , Cicloeximida/farmacologia , Elementos Facilitadores Genéticos , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-jun/genética , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Células Tumorais Cultivadas
8.
J Biol Chem ; 269(52): 32963-71, 1994 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-7806526

RESUMO

TAP-1 (T-cell AP-1) is a previously identified DNA-binding activity that is rapidly induced in activated T cells in the absence of protein synthesis. This activity has been purified over 2,000-fold from the T-cell line MLA144. Purified TAP-1 is a multicomponent complex composed of 38-kDa and 43-kDa junD polypeptides in association with a separate factor(s), distinct from fos, that partly dissociate from the complex during affinity purification but is required for full TAP-1 DNA-binding activity. When reconstituted with TAP-1, this partly dissociated component strongly enhances the DNA-binding activity of the TAP-1 complex. UV-cross-linking analysis identifies the dissociable component of the TAP-1 complex as a separate class of low molecular mass (23-29-kDa) DNA-binding polypeptide(s). 23-29-kDa polypeptides have been partially purified from nuclear extracts derived from MLA144 that enhance TAP-1 DNA-binding activity over 100-fold and increase its contacts with flanking DNA sequence. These results define TAP-1 as a distinct AP-1.junD-containing complex in T cells whose DNA-binding activity is regulated by the interaction of distinct and separate cellular factor(s).


Assuntos
Fatores Biológicos/metabolismo , DNA/metabolismo , Proteínas Proto-Oncogênicas c-jun/isolamento & purificação , Linfócitos T/metabolismo , Fator de Transcrição AP-1/isolamento & purificação , Animais , Sequência de Bases , Hylobates , Dados de Sequência Molecular , Peso Molecular , Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Células Tumorais Cultivadas , Raios Ultravioleta
9.
EMBO J ; 19(15): 4064-73, 2000 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-10921887

RESUMO

Vascular endothelial growth factor (VEGF) has two highly homologous tyrosine kinase receptors: Flt-1 (VEGFR-1) and KDR (VEGFR-2). KDR is strongly phosphorylated on tyrosines and can transmit mitogenic and motogenic signals following VEGF binding, while Flt-1 is markedly less effective in mediating such functions. To dissect the regions that account for the differences between the two receptors, we generated a series of chimeric Flt-1-KDR molecules. We found that the juxtamembrane region of Flt-1 prevents key signaling functions. When the juxtamembrane region of Flt-1 is replaced by that of KDR, Flt-1 becomes competent to mediate endothelial cell migration and phosphatidylinositol 3'-kinase activation in response to VEGF. Further mutational analysis shows that a short divergent sequence is responsible for such repressor function. However, mutant Flt-1 receptors lacking this sequence do not transmit effective proliferative signals, suggesting that this receptor function is regulated separately. These results define a novel functional domain that serves to repress Flt-1 activity in endothelial cells.


Assuntos
Movimento Celular , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/fisiologia , Linfocinas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Sequência de Aminoácidos , Animais , Cromonas/farmacologia , Análise Mutacional de DNA , Ativação Enzimática , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Suínos , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
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