RESUMO
Delta opioid receptors (DOR) are G-protein coupled 7-transmembrane receptors (GPCR), expressed by thymic and splenic T cells, that modulate interleukin (IL)-2 production and proliferation in response to concanavalin A or crosslinking the TCR. Mitogen-activated protein kinases (MAPKs) are involved in mediating intracellular responses to TCR crosslinking. In addition, MAPKs can be activated by signaling cascades that are initiated by the release of G-proteins from GPCRs. To determine whether DORs expressed by T cells signal through the MAPKs, extracellular-regulated kinases (ERKs) 1 and 2, two delta opioid peptides, deltorphin and [D-Ala2,D-Leu5]-enkephalin (DADLE), were studied in Jurkat cells that had been stably transfected with DOR (DOR-Ju.1). These peptides rapidly and dose-dependently induced ERK phosphorylation; pretreatment with naltrindole (NTI), a selective DOR antagonist, abolished this. Pertussis toxin (PTX) also inhibited phosphorylation, indicating the involvement of the Gi/o proteins. Herbimycin A, a protein tyrosine kinase (PTK) inhibitor, reduced the DADLE-induced ERK phosphorylation by 68%. ERK phosphorylation was inhibited by Bisindolylmaleimide 1 (GF109203X), an inhibitor of PKC, and by pretreatment with PMA prior to DADLE. A GTP/GDP exchange assay was used to assess the potential role of Ras in the pathway leading to ERK phosphorylation; DADLE failed to stimulate GTP/GDP exchange in comparison to PMA. Additional studies showed that DADLE stimulated an increase in cfos mRNA; this was reduced by the inhibitor of MAPK/ERK kinase (MEK), PD98059. Therefore, in DOR-Ju.1 cells, DOR agonists stimulate ERK phosphorylation in a Ras independent and PKC-dependent manner; PTKs appear to be involved. MAPKs mediate the increase in cfos mRNA induced by DOR agonists.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Jurkat/química , Proteínas Quinases Ativadas por Mitógeno , Proteínas/metabolismo , Receptores Opioides delta/genética , Proteínas ras/metabolismo , Adjuvantes Imunológicos/metabolismo , Animais , Benzoquinonas , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Carcinógenos/farmacologia , Leucina Encefalina-2-Alanina/farmacologia , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Proteínas Ativadoras de GTPase , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Células Jurkat/enzimologia , Células Jurkat/imunologia , Lactamas Macrocíclicas , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Oligopeptídeos/farmacologia , Toxina Pertussis , Fosforilação , Proteínas Proto-Oncogênicas c-fos/genética , Quinonas/farmacologia , RNA Mensageiro/análise , Receptores Opioides delta/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rifabutina/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Virulência de Bordetella/farmacologia , Proteínas Ativadoras de ras GTPaseRESUMO
Although an immunosuppressant, FK506, has been known to stimulate growth hormone (GH) release from rat somatotropes, the cellular signaling mechanism is unknown. In the present study, intracellular signaling pathways were investigated for FK506- and cyclosporin A (CsA)-induced GH release in cultured rat anterior pituitary cells. Northern and Western blot analysis revealed that the FK506-binding protein (FKBP12) and the CsA-binding protein (cyclophilin A) exist at the mRNA and protein level in the rat anterior pituitary tissue. FK506 and CsA increased GH release in a dose-dependent manner and inhibited calcineurin (CaN) activity in the cultured pituitary cells. The third immunosuppressant, rapamycin (RP), inhibited the FK506-induced GH release, although RP alone had no effect. Protein kinase A (PKA) inhibitors, H-89 and HA-1004 and EGTA blocked FK506- and CsA-induced GH release. TGF-beta did not alter basal GH release, but inhibited FK506-induced GH release. GH primary transcripts were increased by FK506, and the effects were blocked by H-89 and HA-1004. These results suggest that the immunosuppressants, FK506 and CsA, stimulate GH release by inhibiting CaN activity which results in the activation of the PKA system in the rat somatotropes. TGF-beta receptors might be involved in FK506-induced GH release as a separate pathway. FK506 also stimulates GH primary transcripts via a PKA-dependent mechanism in a manner similar to its effects on GH release.
Assuntos
Ciclosporina/farmacologia , Hormônio do Crescimento/metabolismo , Imunossupressores/farmacologia , Transdução de Sinais/imunologia , Sulfonamidas , Tacrolimo/farmacologia , Animais , Northern Blotting , Western Blotting , Tronco Encefálico/química , Tronco Encefálico/citologia , Tronco Encefálico/enzimologia , Calcineurina/metabolismo , Cerebelo/química , Cerebelo/citologia , Cerebelo/enzimologia , Córtex Cerebral/química , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Hormônio do Crescimento/genética , Hormônio do Crescimento/imunologia , Humanos , Hipotálamo/química , Hipotálamo/citologia , Hipotálamo/enzimologia , Imunofilinas/análise , Imunofilinas/genética , Isoquinolinas/farmacologia , Células Jurkat/química , Células Jurkat/enzimologia , Masculino , Peptidilprolil Isomerase/análise , Peptidilprolil Isomerase/genética , Hipófise/química , Hipófise/citologia , Hipófise/imunologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Sirolimo/farmacologia , Proteínas de Ligação a Tacrolimo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/imunologiaRESUMO
Despite extensive study, several of the major components involved in T cell receptor-mediated signaling remain unidentified. Here we report the cloning of the cDNA for a highly tyrosine-phosphorylated 36-38 kDa protein, previously characterized by its association with Grb2, phospholipase C-gamma1, and the p85 subunit of phosphoinositide 3-kinase. Deduced amino acid sequence identifies a novel integral membrane protein containing multiple potential tyrosine phosphorylation sites. We show that this protein is phosphorylated by ZAP-70/Syk protein tyrosine kinases leading to recruitment of multiple signaling molecules. Its function is demonstrated by inhibition of T cell activation following overexpression of a mutant form lacking critical tyrosine residues. Therefore, we propose to name the molecule LAT-linker for activation of T cells.