RESUMO
Using a multistep polymerase chain reaction method, we have produced a construct in which a cDNA sequence encoding the extracellular domain of the human 55-kD tumor necrosis factor (TNF) receptor is attached to a sequence encoding the Fc portion and hinge region of a mouse IgG1 heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. A secreted protein, capable of binding TNF and inactivating it, was produced by the transfected cells. Molecular characterization revealed that this soluble version of the TNF receptor was dimeric. Moreover, the protein could be quantitatively cleaved by treatment with thrombin. However, the monovalent extracellular domain prepared in this way has a greatly reduced TNF inhibitory activity compared with that of the bivalent inhibitor. Perhaps because of its high affinity for TNF, the chimeric protein is far more effective as a TNF inhibitor than are neutralizing monoclonal antibodies. This molecule may prove very useful as a reagent for the antagonism and assay of TNF and lymphotoxin from diverse species in health and disease, and as a means of deciphering the exact mechanism through which TNF interacts with the 55-kD receptor.
Assuntos
Imunoglobulina G/fisiologia , Cadeias Pesadas de Imunoglobulinas/fisiologia , Receptores de Superfície Celular/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Células CHO , Cromatografia de Afinidade , Cricetinae , Humanos , Dados de Sequência Molecular , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/isolamento & purificação , Trombina/farmacologia , Células Tumorais CultivadasRESUMO
Different vectors were constructed that expressed the human interferon-beta (IFN-beta) mRNA constitutively and contained various deletions in the 3' untranslated region (UTR). AU-rich sequences in the 3' UTR were specifically deleted in two vectors. Cell lines secreting human IFN-beta were established by transfecting murine L929 cells with the vectors. These cells showed similar levels of IFN-beta mRNA and secreted comparable amounts of IFN-beta, indicating that the deletion of AU-rich sequences had no effect on the stability and little effect on the efficiency of translation of this mRNA. The synthetic glucocorticoid dexamethasone was previously shown to increase the turnover of IFN-beta mRNA. This activity of dexamethasone was clearly observed only in cells expressing IFN-beta mRNA with AU-rich sequences in the 3' UTR. The increased turnover of this mRNA occurred in the presence of cycloheximide; therefore, it did not require synthesis of new proteins. These findings suggest that glucocorticoids may activate a ribonuclease that degrades mRNAs containing AU-rich sequences in the 3' UTR.
Assuntos
Glucocorticoides/fisiologia , Interferon Tipo I/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Adenina , Northern Blotting , Deleção Cromossômica , Clonagem Molecular , Cicloeximida/farmacologia , DNA/isolamento & purificação , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Interferon Tipo I/metabolismo , Células L , Plasmídeos , RNA Mensageiro/biossíntese , Transfecção , UracilaRESUMO
The ability of morphine to alleviate pain is mediated through a heterotrimeric guanine nucleotide binding protein (G protein)-coupled heptahelical receptor (GPCR), the mu opioid receptor (muOR). The efficiency of GPCR signaling is tightly regulated and ultimately limited by the coordinated phosphorylation of the receptors by specific GPCR kinases and the subsequent interaction of the phosphorylated receptors with beta-arrestin 1 and beta-arrestin 2. Functional deletion of the beta-arrestin 2 gene in mice resulted in remarkable potentiation and prolongation of the analgesic effect of morphine, suggesting that muOR desensitization was impaired. These results provide evidence in vivo for the physiological importance of beta-arrestin 2 in regulating the function of a specific GPCR, the muOR. Moreover, they suggest that inhibition of beta-arrestin 2 function might lead to enhanced analgesic effectiveness of morphine and provide potential new avenues for the study and treatment of pain, narcotic tolerance, and dependence.
Assuntos
Analgésicos Opioides/farmacologia , Arrestinas/fisiologia , Morfina/farmacologia , Receptores Opioides mu/metabolismo , Analgesia , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/metabolismo , Animais , Arrestinas/genética , Sítios de Ligação , Temperatura Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/administração & dosagem , Morfina/metabolismo , Naloxona/metabolismo , Naloxona/farmacologia , Antagonistas de Entorpecentes/metabolismo , Antagonistas de Entorpecentes/farmacologia , Medição da Dor , Limiar da Dor , Fosforilação , Transdução de Sinais , beta-Arrestina 1 , beta-Arrestina 2 , beta-ArrestinasRESUMO
Our laboratory has been testing the hypothesis that genetic modulation of the beta-adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human beta2-adrenergic receptor (beta2AR) or an inhibitor of the beta-adrenergic receptor kinase (betaARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the beta2AR (Adeno-beta2AR) or a peptide betaARK inhibitor (consisting of the carboxyl terminus of betaARK1, Adeno-betaARKct) and tested their ability to potentiate beta-adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-beta2AR infection led to approximately 20-fold overexpression of beta-adrenergic receptors. Protein immunoblots demonstrated the presence of the Adeno-betaARKct transgene. Both transgenes significantly increased isoproterenol-stimulated cAMP as compared to myocytes infected with an adenovirus encoding beta-galactosidase (Adeno-betaGal) but did not affect the sarcolemmal adenylyl cyclase response to Forskolin or NaF. beta-Adrenergic agonist-induced desensitization was significantly inhibited in Adeno-betaARKct-infected myocytes (16+/-2%) as compared to Adeno-betaGal-infected myocytes (37+/-1%, P < 0.001). We conclude that recombinant adenoviral gene transfer of the beta2AR or an inhibitor of betaARK-mediated desensitization can potentiate beta-adrenergic signaling.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Ventrículos do Coração/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Adenilil Ciclases/análise , Agonistas Adrenérgicos beta/farmacologia , Animais , Sobrevivência Celular , Células Cultivadas , AMP Cíclico/metabolismo , Vetores Genéticos , Ventrículos do Coração/citologia , Humanos , Isoproterenol/farmacologia , Masculino , Coelhos , Sarcolema/enzimologia , Transdução de Sinais , TransgenesRESUMO
BACKGROUND: Neointimal hyperplasia involves activation of smooth muscle cells (SMCs) by several G protein-coupled receptor (GPCR) agonists, including endothelin-1, angiotensin II, thrombin, and thromboxane A(2). Signaling of many GPCRs is diminished by GPCR kinase-2 (GRK2). We therefore tested whether overexpression of GRK2 in SMCs could diminish mitogenic signaling elicited by agonists implicated in the pathogenesis of neointimal hyperplasia. METHODS AND RESULTS: Overexpression of GRK2 was achieved in primary rabbit aortic SMCs with a recombinant adenovirus. Control SMCs were infected with an empty vector adenovirus. Inositol phosphate responses to endothelin-1, angiotensin II, thrombin agonist peptide, and platelet-derived growth factor (PDGF) were attenuated by 37% to 72% in GRK2-overexpressing cells (P<0.01), but the response to the thromboxane A(2) analogue U46619 was unaffected. GRK2 also inhibited SMC [(3)H]thymidine incorporation stimulated not only by these agonists (by 30% to 60%, P<0.01) but also by 10% FBS (by 35%, P<0. 05). However, GRK2 overexpression had no effect on epidermal growth factor-induced [(3)H]thymidine incorporation. Agonist-induced tyrosine phosphorylation of the PDGF-beta receptor, but not the epidermal growth factor receptor, was reduced in GRK2-overexpressing SMCs. GRK2 overexpression also reduced SMC proliferation in response to endothelin-1, PDGF, and 10% FBS by 62%, 51%, and 29%, respectively (P<0.01), without any effect on SMC apoptosis. CONCLUSIONS: GRK2 overexpression diminishes SMC mitogenic signaling and proliferation stimulated by PDGF or agonists for several GPCRs. Gene transfer of GRK2 may therefore be therapeutically useful for neointimal hyperplasia.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Mitógenos/fisiologia , Músculo Liso Vascular/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Divisão Celular/fisiologia , Hidrólise , Masculino , Músculo Liso Vascular/citologia , Fosfatidilinositóis/metabolismo , Coelhos , Transdução de Sinais/fisiologia , Timidina/metabolismo , Quinases de Receptores Adrenérgicos betaRESUMO
Recent transgenic as well as gene-targeted animal models have greatly increased our understanding of the molecular mechanisms of normal and compromised heart function. These studies have raised the possibility of using somatic gene transfer as a means for improving cardiac function. DNA transfer to a significant portion of the myocardium has thus far been difficult to accomplish. This review describes current efforts to achieve myocardial gene transfer in several model systems, with particular emphasis placed on adenovirus-mediated gene delivery, its possibilities, and current limitations. (Trend Cardiovasc Med 1997;7:145-150). © 1997, Elsevier Science Inc.
RESUMO
We have exploited the extremely high binding specificity of the 55 kDa human tumor necrosis factor (TNF) receptor in an assay designed to detect TNF with sensitivity limited only by limits in the detection of 131I. Bivalent derivatives of the 55 kDa TNF receptor (referred to here as the TNF binding protein), in which the extracellular domain is coupled to an IgG heavy chain, ordinarily bind TNF with very high affinity as a result of the fact that they interact with two separate sites on the trimer surface. The TNF binding protein is radioiodinated to a high specific activity and then added to plasma at a saturating concentration, so that it binds all active TNF present in the solution. Covalent adducts between molecules of TNF and molecules of the binding protein are then produced by crosslinking with disuccinimidyl suberate (DSS). The complexes are swept out of solution using sepharose beads to which polyclonal anti-TNF antibodies have been affixed. On electrophoresis, the complex presents itself as a band of M(r) = 200 kDa (as distinct from the uncomplexed binding protein, which has a size of 120 kDa and which in any case is removed by washing). As little as 50 fg of active TNF (600,000 trimers) can be detected in a 5 ml sample of plasma using this approach, corresponding to the detection of TNF at a 200 aM concentration. Notably, no TNF is detectable in normal plasma specimens, indicating that normal plasma contains active TNF at a concentration beneath 200 aM.
Assuntos
Receptores do Fator de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/análise , Animais , Células CHO , Cricetinae , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoglobulina G/imunologia , Técnicas Imunológicas , Radioisótopos do Iodo , Peso Molecular , Coelhos , Proteínas Recombinantes/análise , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/imunologiaRESUMO
PURPOSE: Drainage of aqueous humor from the human eye appears dependent on intracellular volume of trabecular meshwork (TM) cells, the predominant cell type of the human outflow pathway. Thus, the modulation of water and solute flux across the plasma membrane of TM cells is predicted to be an important factor in regulating outflow facility. Aquaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations in fluid-secreting and -absorbing tissues. AQP1 is present in many tissues of the human eye, including the TM; however, its role in outflow facility is unknown. The purpose of the present study was twofold: to evaluate the prospect of manipulating AQP1 protein levels in TM cells using sense and antisense mRNA and to investigate the functional role of AQP1 in TM cells. METHODS: An adenovirus (AV) expression system was used to alter AQP1 protein levels. AQP1 protein expression was monitored using immunoblot analysis, and resting cell volume was measured by forward light scatter, electronic cell sizing, and [(14)C]-sucrose/urea equilibration. Permeability of TM monolayers to [(14)C]-sucrose was also assessed as an indirect evaluation of cell volume. RESULTS: AV-mediated gene transfer of AQP1 cDNA to TM cells resulted in a titer-dependent increase in recombinant AQP1, whereas transfer of antisense cDNA decreased native AQP1 protein by 71.7% +/- 5.5% (P < 0.01) after 5 days. A novel finding of this study is that mean resting volumes of AQP1(s) AV-infected TM cells in suspension were 8.7% +/- 3.0% greater (P < 0.05) than control cells. Conversely, AQP1 antisense (as) AV-infected cells had resting volumes 7.8% +/- 2.9% less than control cells (P < 0.05). Similar effects of AQP1 expression on resting cell volume were observed in TM monolayers. Consistent with this finding, paracellular permeability of AQP1(s) AV-infected TM monolayers to [(14)C]-sucrose decreased by 8.0% +/- 1.4% (P < 0.001). CONCLUSIONS: In addition to influencing the osmotic permeability of TM plasma membranes, the level of AQP1 protein expression influences resting intracellular volume and thus paracellular permeability of TM cell monolayers in vitro. These data suggest that AQP1 expression may affect outflow facility in vivo.
Assuntos
Aquaporinas/fisiologia , Malha Trabecular/citologia , Adenovírus Humanos/genética , Adolescente , Idoso , Aquaporina 1 , Antígenos de Grupos Sanguíneos , Permeabilidade da Membrana Celular , Tamanho Celular/fisiologia , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Immunoblotting , Recém-Nascido , Pessoa de Meia-Idade , RNA/isolamento & purificação , RNA Mensageiro/biossíntese , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Malha Trabecular/metabolismoRESUMO
OBJECTIVE: The ability to transfer genes to adult myocardium may have therapeutic implications for cardiac transplantation. We investigated the feasibility of adenovirus-mediated transfer of marker genes LacZ and Luciferase, as well as the potentially therapeutic gene of the human beta2-adrenergic receptor in a rat heterotopic heart transplant model. METHODS: Donor hearts were flushed with 10(12) total viral particles of one of three transgenes. Hearts were harvested at various time points after transplantation. LacZ-treated hearts were assessed by histologic staining and Luciferase-treated hearts were assayed for specific luminescence activity. Hearts treated with beta2-adrenergic receptor underwent radioligand binding assays and immunohistochemistry with the use of an antibody specific for the human beta2-adrenergic receptor. RESULTS: LacZ hearts revealed diffuse myocyte staining as opposed to none within controls at 5 days. Luciferase hearts demonstrated a mean activity of 970,000 +/- 220,000 arbitrary light units versus 500 +/- 200 for the controls (p = 0.001). Total beta2-adrenergic receptor densities (fmol/mg membrane protein) for hearts that received the beta2-adrenergic receptor transgene at 3, 5, 7, 10, and 14 days after infection were as follows: right ventricle, 488.5 +/- 126.8, 519.4 +/- 81.8,* 477.1 +/- 51.8,* 183.0 +/- 6.5,* and 82.7 +/- 19.1; left ventricle, 511.0 +/- 167.6, 1206.4 +/- 321.8,* 525.3 +/- 188.7, 183.5 +/- 18.6,* and 75.9 +/- 15.2 (*p < 0.05 vs control value of 75.6 +/- 6.4). Immunohistochemical analysis revealed diffuse staining of varying intensity within myocardial sarcolemmal membranes. CONCLUSIONS: We conclude that global overexpression of different transgenes is possible during cardiac transplantation and, ultimately, adenovirus-mediated gene transfer may provide a unique opportunity for genetic manipulation of the donor organ, potentially enhancing its function.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Transplante de Coração , Miocárdio/metabolismo , Animais , Modelos Animais de Doenças , Estudos de Viabilidade , Expressão Gênica , Imuno-Histoquímica , Óperon Lac , Luciferases/genética , Luciferases/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Transplante Heterotópico , beta-Galactosidase/metabolismoRESUMO
Tumor necrosis factor (TNF) is a protein hormone which mediates diverse inflammatory conditions, but which also may be involved in physiologic processes. To detect the expression of TNF as it might occur in normal tissues we developed a transgenic mouse line bearing a reporter gene construct in which the INF coding sequence and introns are replaced by a chloramphenicol acetyl transferase (CAT) coding sequence. In these animals, expression of CAT within tissues has been shown to reflect TNF production. We now report upon the pattern of CAT expression that is observed during normal pregnancy. CAT is constitutively expressed in both the fetal and maternal thymuses, and in the placenta, but in no other tissues. Placental CAT activity first becomes measurable at day 13 of gestation, peaks at day 16, and is maintained at high levels until parturition. Crosses between transgenic and non-transgenic mice clearly indicate that the trophoblast, rather than the decidua or uterus, is the source of CAT activity. A soluble TNF receptor/IgG heavy chain chimeric protein, which strongly inhibits TNF activity in vitro and in vivo, was shown to cross the placenta, gaining access to the fetal circulation when administered on the maternal side. However, the chimeric protein did not interrupt pregnancy, and had no obvious effect on fetal development, suggesting that TNF may not be required for completion of a normal gestation.
Assuntos
Prenhez/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Cloranfenicol O-Acetiltransferase/genética , Feminino , Feto/metabolismo , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Placenta/metabolismo , Gravidez , Prenhez/genética , Distribuição Tecidual , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genéticaRESUMO
The pathway of degradation of human interferon-beta (IFN-beta) mRNA was examined in murine C127 cells that carry an expression vector for this mRNA. The IFN-beta mRNA decayed with a half-life of approximately 45 min in actinomycin D-treated cells and became gradually shorter. This mRNA was superinduced in cycloheximide-treated cells, but it also became gradually shorter. However, apparently full-length species of IFN-beta mRNA accumulated after prolonged incubation with cycloheximide. The shortened IFN-beta mRNA species were partially deadenylated and less stable than full-length species. These findings suggest that at least two nuclease activities are involved in degrading IFN-beta mRNA; one deadenylates this mRNA and decays in cycloheximide-treated cells, while the other apparently breaks down deadenylated mRNA.
Assuntos
Interferon gama/genética , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Humanos , Camundongos , Poli A/metabolismo , RNA/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , TransfecçãoRESUMO
Tumor necrosis factor (TNF) induces the synthesis of two proteins of Mr 42 and 36 kDa in human fibroblasts and SK-MEL-109 melanoma cells. To identify these proteins, a lambda gt10 cDNA library was prepared from the mRNA of TNF-treated SK-MEL-109 cells. By screening this library, we found a cDNA that preferentially hybridized to TNF-induced RNA. Hybrid-selected mRNA was translated into a protein of 42 kDa; cDNA sequence analysis followed by a comparison with other known protein sequences identified this protein with plasminogen activator inhibitor, type-2 (PAI-2). After removal of TNF, PAI-2 mRNA turned over rapidly, with an apparent half-life of approximately 2.5 h. Addition of dexamethasone increased the turnover of this mRNA, suggesting that the level of PAI-2 mRNA could be regulated post-transcriptionally by glucocorticoids. PAI-2 was not secreted, but accumulated in fibroblasts continuously treated with TNF.
Assuntos
Fibroblastos/metabolismo , Melanoma/patologia , Inativadores de Plasminogênio/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/patologia , Clonagem Molecular , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Fibroblastos/efeitos dos fármacos , Humanos , Melanoma/genética , Melanoma/metabolismo , Hibridização de Ácido Nucleico , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
A chimeric protein capable of binding and neutralizing tumor necrosis factor (TNF) and lymphotoxin was expressed in mice transduced with a replication-incompetent adenoviral vector into which a TNF inhibitor gene had been engineered. Within 3 days following the injection of 10(9) infectious particles, the TNF inhibitor concentration exceeded 1 mg/ml of plasma; this level of expression was maintained for at least 4 weeks, and detectable TNF inhibitory activity was measured 6 weeks after injection of the recombinant virus. Introduction of the artificial gene produced a phenotypic effect comparable to homozygous deletion of the 55-kDa TNF receptor, in that animals were rendered highly susceptible to infection by Listeria monocytogenes, whereas control animals receiving a replication-incompetent virus coding for beta-galactosidase were capable of resisting Listeria challenge. Adenovirus-mediated transfer of a gene encoding a TNF inhibitor offers a practical means of imposing effective, long-term blockade of TNF activity in vivo for investigational and therapeutic purposes.
Assuntos
Listeriose/imunologia , Receptores do Fator de Necrose Tumoral/química , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adenoviridae/genética , Animais , Técnicas de Transferência de Genes , Imunoglobulina G/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
We previously reported that a chimeric protein consisting of the human p55 TNF receptor covalently linked to a murine IgG1 Fc heavy chain acts as an efficient TNF inhibitor, as a result of its high binding affinity for native TNF trimers of both murine and human origin. A transgenic mouse line constitutively expressing the inhibitor from a cytomegalovirus promoter was established. All organs examined expressed the transgene. TNF inhibitory activity was easily detected in plasma of transgenic animals but not in plasma of nontransgenic littermates. This founder line was not found to have any obvious phenotype. Specifically, transgenic mice born to wild-type or transgenic females were indistinguishable from nontransgenic littermates with respect to their size at birth, at three months and at six months of age, with respect to the size and morphology of their lymphoid organs and with respect to their hematocrit, white blood cell count, and differential. Although TNF is known to be constitutively expressed by cells of the thymus and trophoblast, the lack of a clear phenotype in association with the constitutive expression of a TNF inhibitor suggests that TNF may be dispensable in early development.
Assuntos
Proteínas Recombinantes de Fusão/biossíntese , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Células CHO , Cricetinae , Feminino , Humanos , Imunoglobulina G/biossíntese , Cadeias Pesadas de Imunoglobulinas/biossíntese , Camundongos , Camundongos Transgênicos , RNA/análise , Receptores do Fator de Necrose Tumoral/biossíntese , Proteínas Recombinantes de Fusão/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
G protein-coupled receptor kinases (GRKs) 2 and 3 (beta-adrenergic receptor kinases 1 and 2 (betaARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wild-type littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTPgammaS and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (-/-) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.
Assuntos
Proteínas Serina-Treonina Quinases , Receptores Proteína Tirosina Quinases/fisiologia , Receptores Odorantes/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Regulação para Baixo , Quinase 3 de Receptor Acoplado a Proteína G , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Receptores Proteína Tirosina Quinases/genética , Regulação para Cima , Quinases de Receptores Adrenérgicos betaRESUMO
Contraction and relaxation of airway smooth muscles is mediated, in part, by G protein-coupled receptors (GPCRs) and dysfunction of these receptors has been implicated in asthma. Phosphorylation of GPCRs, by G protein-coupled receptor kinase (GRK), is an important mechanism involved in the dampening of GPCR signaling. To determine whether this mechanism might play a role in airway smooth muscle physiology, we examined the airway pressure time index and heart rate (HR) responses to intravenous administration of the cholinergic agonist methacholine (MCh) in genetically altered mice lacking one copy of GRK2 (GRK2 +/-), homozygous GRK3 knockout (GRK3 -/-), and wild-type littermates. (GRK2 -/- mice die in utero.) GRK3 -/- mice demonstrated a significant enhancement in the airway response to 100 and 250 microgram/kg doses of MCh compared with wild-type and GRK2 +/- mice. GRK3 -/- mice also displayed an enhanced sensitivity of the airway smooth muscle response to MCh. In addition, GRK3 -/- mice displayed an altered HR recovery from MCh-induced bradycardia. Although direct stimulation of cardiac muscarinic receptors measured as vagal stimulation-induced bradycardia was similar in GRK3 -/- and wild-type mice, the baroreflex increase in HR associated with sodium nitroprusside-induced hypotension was significantly greater in GRK3 -/- than wild-type mice. Therefore, these data demonstrate that in the mouse, GRK3 may be involved in modulating the cholinergic response of airway smooth muscle and in regulating the chronotropic component of the baroreceptor reflex.
Assuntos
Brônquios/fisiopatologia , Frequência Cardíaca/fisiologia , Proteínas Serina-Treonina Quinases , Receptores Proteína Tirosina Quinases/deficiência , Antagonistas Adrenérgicos beta/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Barorreflexo/efeitos dos fármacos , Barorreflexo/fisiologia , Brônquios/efeitos dos fármacos , Broncoconstritores/farmacologia , Estimulação Elétrica , Quinase 3 de Receptor Acoplado a Proteína G , Frequência Cardíaca/efeitos dos fármacos , Injeções Intravenosas , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Knockout/genética , Camundongos Transgênicos/genética , Nitroprussiato/farmacologia , Propranolol/farmacologia , Receptores Proteína Tirosina Quinases/genética , Nervo Vago/fisiologiaRESUMO
Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring beta-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular beta-adrenergic signaling defects including down-regulation of myocardial beta-adrenergic receptors (beta-ARs), functional beta-AR uncoupling, and an up-regulation of the beta-AR kinase (betaARK1). Adenoviral-mediated gene transfer of the human beta2-AR or an inhibitor of betaARK1 to these failing myocytes led to the restoration of beta-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of betaARK1 activity in the heart.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Técnicas de Transferência de Genes , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/fisiopatologia , Receptores Adrenérgicos beta/genética , Transdução de Sinais/genética , Adenoviridae/genética , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos , Ventrículos do Coração/citologia , Isoproterenol/farmacologia , Masculino , Coelhos , Receptores Adrenérgicos beta/biossíntese , Taquicardia , Transgenes , Quinases de Receptores Adrenérgicos betaRESUMO
Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.
Assuntos
Anticorpos Monoclonais , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Membrana , Proteínas Serina-Treonina Quinases , Receptores de Superfície Celular/biossíntese , Receptores Acoplados a Proteínas G , Angiotensina II/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Linhagem Celular , Células Cultivadas , Galinhas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , Quinase 3 de Receptor Acoplado a Proteína G , Quinases de Receptores Acoplados a Proteína G , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/isolamento & purificação , Coração/efeitos dos fármacos , Ventrículos do Coração , Humanos , Immunoblotting , Isoproterenol/farmacologia , Cinética , Miocárdio/metabolismo , Neurônios Aferentes/enzimologia , Neurônios Aferentes/fisiologia , Fosforilação , Coelhos , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/isolamento & purificação , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/isolamento & purificação , Transfecção , Quinases de Receptores Adrenérgicos betaRESUMO
This study was designed to determine if adenoviral-mediated delivery of a transgene encoding the beta 2-adrenergic receptor (beta 2-AR) to the carotid arterial wall could result in alterations in in vivo vascular function. De-endothelialized rat carotid arteries were infused in vivo with 0.1 mg/ml elastase and adenovirus [6 x 10(9) plaque forming units (PFU)] containing either the marker gene beta-galactosidase (Adeno-beta-gal), DNA encoding the human beta 2-AR (Adeno-beta 2-AR), or no transgene. This low concentration of elastase increased the water permeability (5.2 +/- 0.6 v 1.9 +/- 0.4 x 10(-8) cm/s/mmHg, n = 4, P < 0.0001) without affecting either the vasomotor responsiveness or the morphology of the arterial wall. A transfection efficiency of 73% was achieved with Adeno-beta-gal (n = 3). beta-gal expression was associated with infrequent appearance of T and B lymphocytes, or neutrophil infiltration. Five days after infection with Adeno-beta 2-AR, the total beta-AR density increased six-fold (67.8 +/- 3.4 v 397.0 +/- 155.5 fmol/mg protein, n = 5, P < 0.01); isoproterenol-induced vasorelaxation at transmural pressures from 10-110/mmHg increased (P < 0.01) compared to arteries exposed to control virus (empty adenovirus), n = 4; and isoproterenol-stimulated cAMP production was increased by 65% (n = 5). Thus, adenoviral-mediated delivery of beta 2-ARs into large artery walls results in enhanced beta-AR-mediated vasorelaxation via augmentation in cAMP levels in vascular smooth muscle cells.