RESUMO
Microdomains have been proposed to explain specificity in the myriad of possible cellular targets of cAMP. Local differences in cAMP levels can be generated by phosphodiesterases, which control the diffusion of cAMP. Here, we address the possibility that adenylyl cyclases, the source of cAMP, can be primary architects of such microdomains. Distinctly regulated adenylyl cyclases often contribute to total cAMP levels in endogenous cellular settings, making it virtually impossible to determine the contribution of a specific isoform. To investigate cAMP dynamics with high precision at the single-isoform level, we developed a targeted version of Epac2-camps, a cAMP sensor, in which the sensor was tagged to a catalytically inactive version of the Ca(2+)-stimulable adenylyl cyclase 8 (AC8). This sensor, and less stringently targeted versions of Epac2-camps, revealed opposite regulation of cAMP synthesis in response to Ca(2+) in GH(3)B(6) pituitary cells. Ca(2+) release triggered by thyrotropin-releasing hormone stimulated the minor endogenous AC8 species. cAMP levels were decreased by inhibition of AC5 and AC6, and simultaneous activation of phosphodiesterases, in different compartments of the same cell. These findings demonstrate the existence of distinct adenylyl-cyclase-centered cAMP microdomains in live cells and open the door to their molecular micro-dissection.
Assuntos
Adenilil Ciclases/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Sondas Moleculares/metabolismo , Isoformas de Proteínas/metabolismo , Adenilil Ciclases/química , Adenilil Ciclases/genética , Sinalização do Cálcio , Domínio Catalítico/genética , Linhagem Celular , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Microdomínios da Membrana , Microscopia de Fluorescência , Sondas Moleculares/genética , Diester Fosfórico Hidrolases/metabolismo , Adeno-Hipófise/citologia , Engenharia de Proteínas , Hormônio Liberador de Tireotropina/metabolismoRESUMO
Capacitative Ca(2+) entry (CCE), which occurs through the plasma membrane as a result of Ca(2+) store depletion, is mediated by stromal interacting molecule 1 (STIM1), a sensor of intracellular Ca(2+) store content, and the pore-forming component Orai1. However, additional factors, such as C-type transient receptor potential (TRPC) channels, may also participate in the CCE apparatus. To explore whether the store-dependent Ca(2+) entry reconstituted by coexpression of Orai1 and STIM1 has the functional properties of CCE, we used the Ca(2+)-calmodulin stimulated adenylyl cyclase type 8 (AC8), which responds selectively to CCE, whereas other modes of Ca(2+) entry, including those activated by arachidonate and the ionophore ionomycin, are ineffective. In addition, the Ca(2+) entry mediated by previous CCE candidates, diacylglycerol-activated TRPC channels, does not activate AC8. Here, we expressed Orai1 and STIM1 in HEK293 cells and saw a robust increment in CCE, and a proportional increase in CCE-stimulated AC8 activity. Inhibitors of the CCE assembly process ablated the effects on cyclase activity in both AC8-overexpressing HEK293 cells and insulin-secreting MIN6 cells endogenously expressing Ca(2+)-sensitive AC isoforms. AC8 is believed to be closely associated with the source of CCE; indeed, not only were AC8, Orai1, and STIM1 colocalized at the plasma membrane but also all three proteins occurred in lipid rafts. Together, our data indicate that Orai1 and STIM1 can be integral components of the cAMP and CCE microdomain associated with adenylyl cyclase type 8.
Assuntos
Adenilil Ciclases/metabolismo , Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Adenilil Ciclases/fisiologia , Animais , Canais de Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Humanos , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Ratos , Molécula 1 de Interação Estromal , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPC/fisiologiaRESUMO
Excessive apoptosis of trophoblast cells is thought to be a contributing factor in complications of pregnancy such as pre-eclampsia. Hepatocyte growth factor (HGF) inhibits apoptosis in trophoblasts and we have investigated the signalling pathways through which this anti-apoptotic effect is mediated. Treatment of cells with HGF led to rapid phosphorylation of Akt while an Akt inhibitor blocked the protective effect of HGF. Glycogen synthase kinase-3beta (GSK-3beta) was found to be one of the downstream targets of Akt. HGF treatment inactivated GSK-3beta which in turn led to the activation of the transcription factor beta-catenin. Pharmacological inhibition of GSK-3beta, independently of HGF treatment, strongly increased both beta-catenin activity and cell survival, suggesting that beta-catenin alone has a pronounced anti-apoptotic effect. We also found that both HGF treatment and pharmacological activation of beta-catenin leads to increased expression of inducible nitric oxide synthase (iNOS). We suggest that the Akt mediated activation of beta-catenin leads to inhibition of trophoblast apoptosis following increased expression of iNOS.
Assuntos
Apoptose , Proteínas do Citoesqueleto/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Óxido Nítrico Sintase/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Trofoblastos/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas I-kappa B/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo II , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Trofoblastos/citologia , Trofoblastos/enzimologia , Regulação para Cima , beta CateninaRESUMO
Adenylyl cyclases (ACs) are a family of critically important signaling molecules that are regulated by multiple pathways. Adenylyl cyclase 8 (AC8) is a Ca(2+) stimulated isoform that displays a selective regulation by capacitative Ca(2+) entry (CCE), the process whereby the entry of Ca(2+) into cells is triggered by the emptying of intracellular stores. This selectivity was believed to be achieved through the localization of AC8 in lipid raft microdomains, along with components of the CCE apparatus. In the present study, we show that an intact leucine zipper motif is required for the efficient N-linked glycosylation of AC8, and that this N-linked glycosylation is important to target AC8 into lipid rafts. Disruption of the leucine zipper by site-directed mutagenesis results in the elimination of N-glycosylated forms and their exclusion from lipid rafts. Mutants of AC8 that cannot be N-glycosylated are not demonstrably associated with rafts, although they can still be regulated by CCE; however, raft integrity is required for the regulation of these mutants. These findings suggest that raft localized proteins in addition to AC8 are needed to mediate its regulation by CCE.
Assuntos
Adenilil Ciclases/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Microdomínios da Membrana/enzimologia , Adenilil Ciclases/química , Adenilil Ciclases/genética , Linhagem Celular , Glicosilação , Humanos , Isoenzimas , Zíper de Leucina , Microdomínios da Membrana/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Mutação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico , Transfecção , Tunicamicina/farmacologiaRESUMO
Preeclampsia complicates 5 to 10% of pregnancies and is a leading cause of maternal and fetal mortality and morbidity. Although the cause is unknown, inadequate invasion and remodeling of maternal uterine arteries by extravillous trophoblasts (EVTs) in the first trimester is a common feature. Uterine spiral artery resistance as detected by Doppler ultrasound is commonly used in the second trimester to identify pregnancies destined to develop preeclampsia. Correlation between high uterine resistance and the failure of trophoblast invasion has been reported as early as 12 weeks. However, the reason for this failure has not been established. Understanding the processes involved would significantly improve our diagnostic potential. In this study, we correlated increased first trimester uterine artery resistance with a biological abnormality in trophoblast function. EVTs derived from high-resistance pregnancies were more sensitive to apoptotic stimuli than those from normal-resistance pregnancies. Survival of EVTs from high-resistance pregnancies could be increased by nitric oxide, whereas inhibition of nitric oxide in cells from normal-resistance pregnancies increased apoptotic sensitivity. This predates the onset of symptoms by several weeks and provides evidence for a mechanism responsible for the incomplete uterine vessel remodeling and the differences in artery resistance between preeclamptic and normal pregnancies.