RESUMO
Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.
Assuntos
Citocinas/farmacologia , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/enzimologia , Linfocinas/farmacologia , Metaloendopeptidases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetinae , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Interleucina-1/farmacologia , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Receptores Proteína Tirosina Quinases/química , Receptor de TIE-1 , Receptores de TIE , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador alfa/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The phototransduction process in cones has been proposed to involve a G protein that couples the signal from light-activated visual pigment to the effector cyclic GMP phosphodiesterase. Previously, we have identified and purified a G beta gamma complex composed of a G beta 3 isoform and an immunochemically distinct G gamma subunit (G gamma 8) from bovine retinal cones (Fung, B. K.-K., Lieberman, B. S., and Lee, R. H. (1992) J. Biol. Chem. 267, 24782-24788; Lee, R. H., Lieberman, B.S., Yamane, H. K., Bok, D., and Fung, B. K.-K. (1992a) J. Biol. Chem. 267, 24776-24781). Based on the partial amino acid sequence of this cone G gamma 8, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding G gamma 8. The cDNA insert of this clone includes an open reading frame of 207 bases encoding a 69-amino acid protein. The predicted protein sequence of G gamma 8 shares a high degree of sequence identity (68%) with the G gamma (G gamma 1) subunit of rod transducin. Similar to rod G gamma 1, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for G gamma 8 is present at a high level in the retina and at a very low level in the lung, but is undetectable in other tissues. Immunostaining of bovine retinal sections with an antipeptide antibody against the N-terminal region of G gamma 8 further shows a differential localization of G gamma 8 to cones with a pattern indistinguishable from that of G beta 3. This finding suggests that G beta 3 gamma 8 is a component of cone transducin involved in cone phototransduction and color vision.
Assuntos
Proteínas de Ligação ao GTP/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA Complementar , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Soros Imunes , Imuno-Histoquímica , Dados de Sequência Molecular , Prenilação de Proteína , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosAssuntos
Alquil e Aril Transferases , Cisteína/análogos & derivados , Diterpenos/síntese química , Proteínas de Ligação ao GTP/metabolismo , Prenilação de Proteína , Sequência de Aminoácidos , Animais , Cisteína/síntese química , Cisteína/metabolismo , Diterpenos/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Farnesiltranstransferase , Proteínas de Ligação ao GTP/isolamento & purificação , Substâncias Macromoleculares , Ácido Mevalônico/metabolismo , Dados de Sequência Molecular , Peso Molecular , Técnica de Diluição de Radioisótopos , Ratos , Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Transferases/metabolismo , TrítioRESUMO
Vertebrate retinal cones play a major role in both photopic vision and color perception. Although the molecular mechanism of visual excitation in the cone is not as well understood as in the rod, it is generally thought to involve a cone-specific G protein (cone transducin) that couples the cone visual pigment to a cGMP phosphodiesterase. Like all other G proteins, cone transducin is most likely a heterotrimer consisting of G alpha, G beta, and G gamma subunits. A G alpha subunit of cone transducin has been localized to the outer segment of bovine cones, but its associated G beta and G gamma subunits are unknown. To identify the G beta subunit involved in the phototransduction process of cones, we have developed a panel of antipeptide antisera against the most diverse region of the amino acid sequences encoded by G beta 1, G beta 2, and G beta 3 cDNAs and used them to determine the distribution of the G beta isoforms in different retinal preparations. We found that the G beta 3 subunit is present in bovine retinal transducin and phosducin-T beta gamma complex preparations which were previously thought to contain only G beta 1. Analysis of its subcellular distribution indicated that G beta 3 is predominantly cytoplasmic. Immunocytochemical staining of bovine retinal sections with the anti-G beta 3 antiserum further revealed a specific localization of G beta 3 in cones but not in rods. In contrast, anti-G beta 1 antiserum stained only the rods. These results suggest that G beta 3 is the G beta subunit of cone transducin and confirms the proposition that rods and cones utilize distinct signaling proteins for phototransduction.
Assuntos
Proteínas de Ligação ao GTP/análise , Células Fotorreceptoras/citologia , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , DNA/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Soros Imunes , Immunoblotting , Imuno-Histoquímica , Substâncias Macromoleculares , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Células Fotorreceptoras/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/química , Retina/fisiologia , Frações Subcelulares/química , Frações Subcelulares/fisiologiaRESUMO
We showed previously that a 23-kDa guanine nucleotide-binding protein (G protein) purified from bovine brain membranes is carboxyl methylated and that this modification occurs at or near the membrane-binding domain. In the present study, we identified this small G protein as G25K (formerly termed Gp). We demonstrated that proteolytic digests of 3H-methylated G25K contained radiolabeled material that coeluted with synthetic S-(geranylgeranyl)cysteine methyl ester on reversed-phase HPLC. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the isoprenoid moiety and carboxyl methyl ester are localized on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G25K by Raney nickel treatment positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. These results suggest that geranylgeranyl modification and perhaps methyl esterification function in the membrane localization of this small G protein.
Assuntos
Encéfalo/metabolismo , Cisteína/análogos & derivados , Diterpenos/análise , Proteínas de Ligação ao GTP/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos , Bovinos , Membrana Celular/metabolismo , Cisteína/análise , Proteínas de Ligação ao GTP/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Metilação , Dados de Sequência Molecular , Peso Molecular , Oligopeptídeos/síntese química , Fragmentos de Peptídeos/isolamento & purificaçãoRESUMO
We have shown previously that guanine nucleotide-binding protein (G protein) beta gamma complexes purified from bovine brain membranes are methyl esterified on a C-terminal cysteine residue of the gamma polypeptide. In the present study, 3H-methylated G beta gamma complexes cleaved to their constituent amino acids by exhaustive proteolysis were shown to contain radiolabeled material that coeluted with geranylgeranylcysteine methyl ester on reversed-phase HPLC and two TLC systems. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the prenyl modification occurs on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G beta gamma by treatment with Raney nickel positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. To delineate the distribution of this modification among gamma subunits, purified G beta gamma complexes were separated into 5-kDa (gamma 5) and 6-kDa (gamma 6) forms of the gamma polypeptide by reversed-phase HPLC. Gas chromatography-coupled mass spectrometry analyses of Raney nickel-treated purified gamma 5 and gamma 6 subunits showed that both polypeptides were modified by geranylgeranylation. These results demonstrate that at least two forms of brain gamma subunit are posttranslationally modified by geranylgeranylation and carboxyl methylation. These modifications may be important for targeting G beta gamma complexes to membranes.
Assuntos
Química Encefálica , Cisteína/análogos & derivados , Diterpenos/análise , Proteínas de Ligação ao GTP/isolamento & purificação , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Cisteína/análise , Cisteína/síntese química , Diterpenos/síntese química , Cromatografia Gasosa-Espectrometria de Massas , Substâncias Macromoleculares , Peso Molecular , Técnica de Diluição de Radioisótopos , Terpenos/síntese química , TrítioRESUMO
The cyclic GMP phosphodiesterase of the retinal rod is composed of three distinct types of polypeptides: alpha (90 kDa), beta (86 kDa), and gamma (10 kDa). The gamma subunit has been shown to inhibit phosphodiesterase activity associated with alpha and beta. To investigate the subunit stoichiometry of the retinal phosphodiesterase, we have developed a panel of monoclonal and peptide antibodies that recognize individual phosphodiesterase subunits. By quantitative and immunochemical analysis of the purified subunits, we have shown that each phosphodiesterase molecule contains one copy each of alpha and beta subunit and two copies of gamma subunit. Moreover, gamma can be chemically cross-linked to both alpha and beta, but not to itself, suggesting that alpha and beta may each bind one gamma. The phosphodiesterase is fully activated when both copies of gamma were removed by proteolysis with trypsin. Upon recombination of the purified gamma subunit with the trypsin-activated phosphodiesterase containing alpha beta, the alpha beta gamma 2 stoichiometry is once again restored, with concomitant total inhibition of activity. Our results suggest that at least two activated transducin molecules are required to fully activate one molecule of phosphodiesterase in retinal rods.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Células Fotorreceptoras/enzimologia , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-GMP Cíclico Fosfodiesterases/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anuros , Sítios de Ligação , Bovinos , Reagentes de Ligações Cruzadas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Camundongos , Conformação Proteica , Coelhos , Tripsina/farmacologiaRESUMO
The gamma polypeptide of brain G-proteins is carboxyl methylated when the purified beta gamma subunit complex is reconstituted with S-adenosyl-[3H-methyl]-L-methionine and a methyltransferase present in detergent-stripped brain membranes. By chromatographic analysis of the 3H-amino acid generated by exhaustive proteolysis and performic acid oxidation of the 3H-methylated beta gamma complex, we show that this modification occurs on the alpha-carboxyl group of a C-terminal cysteine residue. Our result suggests that brain G-protein may undergo multiple covalent modification steps, including proteolytic removal of the three terminal amino acids from the predicted common C-terminal Cys-Xaa-Xaa-Xaa sequence, and the methyl esterification of the resulting terminal cysteine residue. This modification is likely to be associated with lipidation at the sulfhydryl group of the same cysteine, which would explain the tight membrane binding property of the brain beta gamma complex.
Assuntos
Química Encefálica , Ácidos Carboxílicos/análise , Cisteína/análise , Ésteres/análise , Proteínas de Ligação ao GTP/isolamento & purificação , Animais , Bovinos , Membrana Celular/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Hidrólise , Lipídeos de Membrana/fisiologia , Metilação , Ligação Proteica , S-AdenosilmetioninaRESUMO
We have purified to homogeneity a 23-kDa protein from bovine brain membranes using [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding as an assay. GTP gamma S binding to the purified protein is inhibited by GDP, GTP, and GTP analogs but not by cGMP, GMP, or adenine nucleotides, consistent with the nucleotide-binding behavior of members of the family of GTP-binding regulatory proteins. On addition of the methyl donor S-adenosyl-L-methionine and a methyltransferase present in bovine brain membranes, the purified 23-kDa G-protein is carboxyl methylated. When subjected to limited tryptic proteolysis, the 23-kDa protein is converted to a 22-kDa major fragment with concomitant release of a carboxyl methylated protein fragment of 1 kDa. Furthermore, when the cleaved protein is reconstituted with stripped bovine brain membranes, the small carboxyl-methylated fragment but not the 22-kDa major fragment is found to reassociate with the membranes. These results indicate that the site of carboxyl methylation and the region responsible for membrane anchoring, most likely, are localized to a small region at the carboxyl terminus. It is attractive to speculate that carboxyl methylation and membrane anchoring are interrelated processes and play key roles in the function of this small G-protein.