GPCRs: Adrenergic receptor gets bent out of shape by morphine

Morphine inhibits adrenergic receptor signaling by inducing a trans-conformational switch in the α_2A-adrenergic receptor (α_2A-AR) when in a complex with the morphine receptor, MOR.

G protein-coupled receptors (GPCRs) are classically thought to signal as monomers. However, recent studies have indicated that GPCRs can also exist as constitutive homo- and hetero-oligomers, which have been shown to modulate signaling to G proteins. In Nature Chemical Biology, Jean-Pierre Vilardaga, Martin Lohse and colleagues now explore the molecular mechanisms that mediate GPCR heterodimer cross-talk and identify direct trans-inhibition as a mechanism for receptor inactivation. They reveal that a complex consisting of norepinephrine (NE)-bound α_2A-adrenergic receptor (α_2A-AR) and μ-opioid receptor (MOR) undergoes a conformational switch upon binding of the MOR agonist morphine. This conformational change decreases both inhibitory G protein (G_i) and MAP kinase activation.

The authors used fluorescence resonance energy transfer (FRET) to verify a previously reported agonist-independent interaction between α_2A-AR and MOR. They also employed a recently developed FRET technique variation that detects real time intramolecular GPCR conformation changes in live cells. Cyan fluorescent protein (CFP) was fused to the C terminus of α_2A-AR, and a tetracysteine motif — which can be targeted by fluorescein arsernical hairpin binder (FlAsH) — was inserted into the intracellular domain of this receptor. The FRET signal from HEK293 cells transfected with MOR and the modified adrenergic receptor demonstrated an NE-induced conformational change in α_2A-AR that is indicative of the receptor's active state. Treatment of the cells with both NE and morphine triggered a further rapid and reversible conformational switch in α_2A-AR.

To determine whether effector signaling properties could be influenced by the MOR–α_2A-AR trans-conformation effect, Vilardaga and colleagues investigated the modulation of G_i activation. They measured FRET changes between yellow fluorescent protein (YFP)-tagged Gα_i and CFP-tagged Gγ₂ in live cells stimulated with both GPCR ligands. Indeed, morphine specifically suppressed NE-induced G_i activation. Further downstream, morphine impaired NE-induced ERK1/2 phosphorylation in a concentration-dependent manner. Thus, activation of the opioid receptor protomer hinders adrenergic receptor-mediated cell signaling.

These findings depict a novel mechanism of rapid GPCR inactivation: the direct isomerization of the α_2A-AR protomer by its activated binding partner, MOR, results in inhibition of adrenergic receptor mediated signaling. It will be interesting to test whether this mechanism applies to other GPCR oligomers and it remains to be seen whether this mode of MOR cross-talk can mediate the analgesic effects of morphine.

Kira Anthony
Pathway Interaction Database

Original Reference:
Vilardaga, J-P., Nikolaev, V. O., Lorenz, K., Ferrandon, S., Zhuang, Z. & Lohse, M. J.
Conformational cross-talk between α_2A-adrenergic and μ-opioid receptors controls cell signaling
Nature Chemical Biology 4, 126-131 (2008).
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Angiogenesis: PGC-1α provides a breath of fresh air

The transcriptional coactivator PGC-1α and transcription factor ERR-α stimulate angiogenesis in hypoxic conditions by directly upregulating VEGF expression.

Ischemia can be countered by neovascularization at the oxygen-deprived site. It has long been known that hypoxia inducible factor (HIF) promotes angiogenesis under hypoxic conditions by upregulating vascular endothelial growth factor (VEGF) and other pro-angiogenic factors. In Nature, Spiegelman and colleagues now document a novel HIF-independent angiogenic pathway where the transcriptional coactivator PGC-1α (peroxisome-proliferator-activated receptor-γ coactivator-1α) upregulates expression of VEGF and other angiogenic factors by stimulating the transcription factor ERR-α (estrogen-related receptor-α).

PGC-1α regulates cellular energy metabolism in muscle by promoting fatty acid oxidation. The authors chose to study PGC-1α activity during the ischemic response because of its known role in cellular respiration. Hypoxic, nutrient-deprived conditions caused a robust upregulation of PGC-1α mRNA and protein in cultured cells. Furthermore, PGC-1α overexpression increased the amount of VEGF, PDGF-BB (platelet-derived growth factor) and angiopoietin 2 mRNA and secreted VEGF protein in hypoxic conditions in cell culture. Depletion of PGC-1α blunted VEGF accumulation of in these conditions.

The link between PGC-1α and pro-angiogenic factors suggested a potential role for PGC-1α in angiogenesis, which was assessed in a mouse model of ischemia. Wild-type mice recover from induced muscle ischemia by inducing angiogenesis. Muscle neovascularization was severely impaired in PGC-1α-deficient mice, but greatly accelerated in mice with targeted overexpression of PGC-1α in skeletal muscle. PGC-1α overexpression in vivo was also accompanied by a marked increase in expression of VEGF, PDGF-BB and angiopoietin 2, suggesting that PGC-1α may mediate angiogenesis in ischemic conditions by upregulating angiogenic factors.

Intriguingly, PGC-1α-mediated induction of VEGF in ischemic conditions appears to be independent of HIF. Exogenous PGC-1α had no effect on HIF-1α transcriptional activity, nor was the promoter of PGC-1α a target for HIF-1α-mediated transcription. However, overexpression of ERR-α, a known effector of PGC-1α, led to a three-fold increase in VEGF mRNA in primary skeletal muscle cells. In addition, ERR-α was required for PGC-1α-mediated induction of VEGF. PGC-1α and ERR-α strongly upregulated transcription of a synthetic VEGF gene construct encompassing five of the 11 predicted ERR-α response elements and could directly bind to these elements in vitro, strongly suggesting that ERR-α can upregulate VEGF in vivo.

These data describe a new pathway for hypoxia-induced angiogenesis in which PGC-1α stimulates ERR-α to induce VEGF transcription. The mechanism by which PGC-1α is induced during hypoxia, and whether PGC-1α can directly upregulate PDGF-BB and angiopoeitin 2 in ischemic conditions is not yet known. Additional studies will be necessary to determine if PGC-1α is important for the vascularization of another well-known hypoxic environment: tumors.

Emily J. Chenette
Signaling Gateway

Original Reference:
Arany Z. et al.
HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1α
Nature 451, 1008-1012 (2008).
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Ethylene signaling: MAPKing new pathways

Two antagonistic MAPK pathways regulate ethylene signaling in plants through differential phosphorylation of the transcription factor EIN3.

The plant hormone ethylene mediates development, differentiation and stress signaling in Arabidopsis thaliana by regulating MAP kinase (MAPK) cascades. In the absence of ethylene, the Raf-like MAPKKK CTR1 (constitutive triple response 1) represses signaling by directly interacting with the ethylene receptor ETR1. Ethylene inhibits CTR1 activity to relieve this repression, resulting in the activation of an unknown MAPK module that promotes EIN3 (ethylene-insensitive 3) transcriptional activity. In Nature, Yoo et al. now uncover the identity of this MAPK module by showing that ethylene activates the MKK9–MPK3/6 MAPK pathway, and describe how ethylene signaling is regulated through differential phosphorylation of EIN3.

The authors performed a genetic screen in CTR1-deficient cells to identify novel MAPK components of the ethylene response pathway. The screen uncovered the kinase MKK9, which directly phosphorylated and activated MPK3/6 in vitro. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) upregulated MPK3/6 in wild-type, but not MKK9-deficient leaves. Furthermore, constitutively active MKK9 induced a phenotype consistent with continuous exposure to ethylene, suggesting that MKK9–MPK3/6 is a critical positive regulator of ethylene signaling.

Transgenic epistasis analysis placed MKK9 downstream of ETR1 and upstream of EIN3 in the ethylene signaling pathway. Furthermore, MPK3/6 directly phosphorylated EIN3 at Thr 174 in vitro. Phosphorylation at this residue in vivo stabilized EIN3 and promoted its nuclear accumulation; in addition, constitutively active MKK9 robustly induced EIN3-responsive genes. Therefore, the MKK9–MPK3/6 axis promotes the response to ethylene by phosphorylating EIN3 at Thr 174, which upregulates its nuclear accumulation and transcriptional activity.

How does CTR1 negatively regulate the ethylene pathway? Constitutively active CTR1 reduced EIN3 protein levels, but had no effect on EIN3 with a mutation at the Thr 592 position. As Thr 592 is a consensus MAPK phosphorylation site, the authors speculate that CTR1 induces phosphorylation at this site, thereby mediating EIN3 degradation. The kinase responsible for phosphorylating Thr 592 in vivo has not yet been identified.

These data suggest that ethylene–ETR1 signaling activates MKK9, which phosphorylates MPK3/6. Active MPK3/6 then phosphorylates EIN3 at Thr 174, stabilizing the protein and inducing transcription of EIN3 target genes. In the absence of ethylene, CTR1 indirectly promotes phosphorylation of Thr 592, resulting in EIN3 degradation. An important future direction of this work will be to elucidate the proteins involved in CTR1-mediated phosphorylation of EIN3 and to clarify how CTR1 inhibits MKK9–MPK3/MPK3 signaling in the absence of ethylene.

Emily J. Chenette
Signaling Gateway

Original Reference:
Yoo, S-D., Cho, Y-H., Tena, G., Xiong, Y. & Sheen, J.
Dual control of nuclear EIN3 by bifurcate MAPK cascades in C₂H₄ signalling
Nature 451, 789-795 (2008).
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Tumor cell invasion: It's not just ROCK 'n' Rho

The kinase PDK1 promotes cancer cell migration and invasion by blocking the interaction between RhoE and ROCK1.

Certain tumor cells invade surrounding tissues using an 'amoeboid' mode of motility, which is characterized by a rounded cell shape and membrane blebbing. This form of migration requires RhoA- and ROCK-mediated contraction of cortical acto-myosin. However, the mechanisms linking RhoA activation to the cytoskeletal rearrangements required for amoeboid cell migration are not fully understood. In Nature Cell Biology, Pinner and Sahai report that PDK1 (3-phosphoinositide-dependent kinase-1) facilitates amoeboid cell migration by binding to ROCK1 (Rho-associated coiled-coil containing kinase 1), thereby competing with the inhibitory ROCK1/RhoE interaction.

To identify new regulators of amoeboid migration, the authors performed chemical and small-interfering (si) RNA-based screens of A375 melanoma cells in three-dimensional matrices, and identified PDK1. Loss of PDK1 disrupted the actin cytoskeleton, caused an elongated morphology and reduced net amoeboid cell motility in vitro and in vivo. In xenografts, depletion of PDK1 did not affect tumor growth, although it did inhibit egress of tumor cells from the vasculature. Compared to control cells, 60% fewer PDK1-deficient cells were able to colonize lungs when injected into the tail veins of mice. Therefore, PDK1 is important for amoeboid cell migration and efficient invasion.

Sahai and colleagues had previously found that RhoA-mediated activation of ROCK elicited phosphorylation and bundling of myosin light chain (MLC) at the cell cortex. A link between PDK1 and RhoA signaling was uncovered by the observation that PDK1 was required for the phosphorylation and proper localization of MLC at the cell cortex. PDK1 deficiency in 3D cultures also caused ROCK1 to mislocalize to the cytoplasm. However, PDK1 does not affect ROCK1 catalytic activity, as purified PDK1 did not alter RhoA-stimulated ROCK phosphorylation of MLC in vitro, and PDK1 kinase activity was not required to restore ROCK1 membrane localization in PDK1-deficient cells.

How, then, does PDK1 regulate ROCK1 activity? The authors found that PDK1 directly bound to ROCK1 and that the PDK1/ROCK1 interaction was necessary, but not sufficient, to target ROCK1 to the plasma membrane. PDK1 binds to ROCK1 in a region that overlaps with the RhoE binding site, and addition of recombinant PDK1 blocked the inhibitory interaction between ROCK1 and RhoE. These observations suggest that PDK1 normally competes with RhoE for binding to ROCK1, and indicate that the morphology defects ascribed to the lack of PDK1 might be due to an increased association of ROCK1 and RhoE. Indeed, reduction of RhoE in PDK1-deficient cells rescued acto-myosin contraction and restored cell motility. Therefore, PDK1 mediates ROCK1 activation independently of its catalytic activity by inhibiting the ROCK1/RhoE interaction and allowing ROCK1 to localize to the plasma membrane. The signaling pathways that regulate PDK1 activation in this context await further study.

Emily J. Chenette
Signaling Gateway

Original Reference:
Pinner, S. & Sahai, E.
PDK1 regulates cancer cell motility by antagonising inhibition of ROCK1 by RhoE
Nature Cell Biology 10, 127-137 (2008).
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Signaling cross-talk: Pyk-ing the pathway for STAT activation

The calcium-dependent kinase Pyk2 coordinates both interferon-α signaling and calcium signaling in the regulation of Jak1 and STAT1 activation.

Cross-talk between seemingly discrete signaling pathways is a universal mechanism of pathway regulation and signal integration. The importance of pathway cross-talk is well appreciated, but understanding the effect of such signal integration depends on identifying proteins that function at the nexus of canonical signaling pathways and evaluating their importance in transducing stimuli. In Nature Immunology, Ivashkiv and colleagues now report that the tyrosine kinase Pyk2 integrates interferon-α (IFN-α) receptor (IFNAR) signaling and calcium-dependent signaling to modulate Janus kinase (Jak)–signal transducer and activator of transcription (STAT) activation and the pro-inflammatory immune response.

Dimerization of STAT1 controls its nuclear translocation and activation and is known to be regulated by serine and tyrosine phosphorylation. Calcium-activated calmodulin kinase (CaMK) phosphorylates STAT1 at Ser 727 in response to interferons; however, the authors found that IFN-α treatment also stimulated STAT1 Tyr 701 phosphorylation in primary human macrophages. Pharmacologic inhibition of calcium signaling blocked IFN-α-mediated STAT1 tyrosine phosphorylation and transcriptional activity, providing evidence for cross-talk between these two pathways. The calcium-dependent kinase Pyk2 was found to function at the interface of the IFN-α and calcium signaling pathways, as IFN-α signaling caused modest activation of Pyk2, which was blocked by pharmacologic inhibition of calcium signaling. These data document a role for Pyk2 in integrating the calcium signaling and IFN-α pathways.

The Jak1–STAT1 pathway is a well-known effector of IFN-α signaling, and previous reports have suggested a linear sequence of IFNAR–Jak1–STAT1 activation. Surprisingly, Pyk2 also appears to regulate IFN-α-mediated Jak1 and STAT1 activation. Chemical and RNAi-mediated inhibition of Pyk2, as well as dominant-negative Pyk2, blocked IFN-α-induced STAT1 tyrosine phosphorylation in human cells. Furthermore, inhibition of CaMK or Pyk2 blocked IFN-α-mediated tyrosine phosphorylation of Jak1, suggesting that calcium signaling and Pyk2 activation may serve to heighten activation of Jak1 and STAT1-directed transcription. Finally, the receptor-coupled adaptor protein DAP12, which has a known role in regulating calcium signaling in immune cells, was found to regulate IFN-α signaling through the calcium–CaMK–Pyk2 pathway. DAP12-deficient mouse macrophages exhibited lower levels of STAT1 phosphorylation following IFN-α stimulation and low basal levels of Pyk2 and CaMKII phosphorylation compared to wild-type cells.

The study by Ivaskiv and colleagues shows that Pyk2 has a central role in modulating Jak1–STAT1 activation in response to IFN-α and defines a novel role for calcium signaling in regulating IFN-α-mediated pathways. IFNAR activation not only promotes Jak1 phosphorylation through a canonical pathway, but also stimulates its phosphorylation via a Pyk2-dependent pathway. The authors speculate that basal DAP12-coupled receptor signaling or calcium signaling is insufficient to promote Jak1 phosphorylation, but instead exists to amplify IFN-α-mediated Jak1 phosphorylation. However, IFN-α signaling does not affect calcium flux, suggesting that the CaMK–Pyk2 pathway is activated by additional unknown factors.

Emily J. Chenette
Signaling Gateway

Original Reference:
Lu Wang, L., Tassiulas, I. et al.
'Tuning' of type I interferon-induced Jak–STAT1 signaling by calcium-dependent kinases in macrophages
Nature Immunology 9, 186-193 (2008).
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