Superoxide dismutase 1 (SOD1) is an abundant copper/zinc enzyme found in the cytoplasm that converts superoxide into hydrogen peroxide and molecular oxygen. Tetrathiomolybdate (ATN-224) has been recently identified as an inhibitor of SOD1 that attenuates FGF-2- and VEGF-mediated phosphorylation of ERK1/2 in endothelial cells. However, the mechanism for this inhibition was not elucidated. Growth factor (GF) signaling elicits an increase in reactive oxygen species (ROS), which inactivates protein tyrosine phosphatases (PTP) by oxidizing an essential cysteine residue in the active site. ATN-224-mediated inhibition of SOD1 in tumor and endothelial cells prevents the formation of sufficiently high levels of H ₂ O ₂ , resulting in the protection of PTPs from H ₂ O ₂ -mediated oxidation. This, in turn, leads to the inhibition of EGF-, IGF-1-, and FGF-2-mediated phosphorylation of ERK1/2. Pretreatment with exogenous H ₂ O ₂ or with the phosphatase inhibitor vanadate abrogates the inhibition of ERK1/2 phosphorylation induced by ATN-224 or SOD1 siRNA treatments. Furthermore, ATN-224-mediated SOD1 inhibition causes the down-regulation of the PDGF receptor. SOD1 inhibition also increases the steady-state levels of superoxide, which induces protein oxidation in A431 cells but, surprisingly, does not oxidize phosphatases. Thus, SOD1 inhibition in A431 tumor cells results in both prooxidant effects caused by the increase in the levels of superoxide and antioxidant effects caused by lowering the levels of H ₂ O ₂ . These results identify SOD1 as a master regulator of GF signaling and as a therapeutic target for the inhibition of angiogenesis and tumor growth.

We sought to determine if different beta-adrenergic receptor (betaAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. Employing enriched nuclear preparations, we assayed the specific presence of betaAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of beta 1AR and beta 3AR but, surprisingly, not the beta 2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear betaAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta 3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another beta 3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta 1AR-selective agonist, xamoterol, nor a beta 2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear betaAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.

Endothelins are thought to act through two specific, plasmalemmal G protein-coupled receptor subtypes, ET_AR and ET_BR. However, in subfractionated cardiac membranes, ET_AR immunoreactivity was detected only in the plasma membrane whereas ET_BR immunoreactivity was detected predominantly in membranes of intracellular origin. Confocal microscopy demonstrated the presence of intracellular ET_AR and ET_BR in ventricular myocytes. ET_AR were primarily on plasma membrane (surface membranes and transverse-tubules) and to a lesser extent on the nucleus while ET_BR localized primarily to the nuclei. Western blot analysis of nuclei isolated from the heart indicated the presence of endothelin receptors: both ET_AR and ET_BR copurified with nucleoporin 62, whereas markers of endoplasmic reticulum and Golgi membranes were depleted. Radioligand binding studies revealed that isolated nuclei contain specific [¹²⁵I]ET-1 binding sites. Specific [¹²⁵I]ET-1 binding was reduced by 70–80% using the ET_AR-selective antagonist BQ610 and 20–30% using the ET_BR-specific antagonist BQ788. IRL-1620, an ET_BR-specific agonist, also reduced [¹²⁵I]ET-1 binding. Furthermore, ET-1 and IRL-1620 altered the incorporation of ³²P into nuclear proteins and caused a transient increase in nuclear Ca²⁺ concentration. Hence, cardiac nuclei possess both ET_AR and ET_BR subtypes, which are functional with respect to ligand binding and are coupled to signaling mechanisms within the nuclear membrane.

Signaling from internalizing and endosomal receptors has almost become a classic GPCR paradigm in the last several years. However, it has become clear in recent years that GPCRs also elicit signals when resident at other subcellular sites including the endoplasmic reticulum, Golgi apparatus, and the nucleus. In this review we discuss the nature, function, and trafficking of nuclear GPCR signaling complexes, as well as potential sources of endogenous and exogenous ligands. Finally, we pose a series of questions that will need to be answered in the coming years to confirm and extend this as a new paradigm for GPCR signaling.

The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 ( Txnrd1 ^−/− ) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1 -depleted cells displayed increased phosphorylation of PDGF-β receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-β receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.