Ligand Sensitivity Research Articles

Hemodynamic consequences of spatially‐dependent sympathetic regulation in skeletal muscle arteriolar trees (678.14)

The sympathetic nervous system (SNS) elicits vasoconstriction on skeletal muscle arterioles via release of neurotransmitters norepinephrine (NE; acting on α1‐ and α2R), neuropeptide Y (NPY; acting on Y1R), and adenosine‐triphosphate (ATP; acting on P2X1R). Previous studies have shown that the level of α‐adrenoreceptor modulation is dependent on vessel branch order. However, to date, no studies have investigated the integrated roles of α1R, α2R, Y1R, and P2X1R modulation on arteriolar network hemodynamics. Thus, we evaluated the effects of phenylephrine (PE; α1R agonist), UK 14,304 (UK; α2R agonist), NPY, and ATP on arteriolar diameters in continuously branching arteriolar trees (1st order to 5th order; 1A‐5A). In male rats, concentration‐response curves (PE and ATP: 10‐9‐10‐4M; UK: 10‐9‐10‐5M; NPY: 10‐13‐10‐8M) of arteriolar diameter changes were evaluated in the gluteus maximus muscle (n蠅5 rats/group). Like others, we observed that α1R and α2R control is greatest at large (1A‐2A) arterioles, with declining sensitivity in subsequent branch orders approaching capillaries. Beyond this, we have observed that NPY and ATP elicit the greatest constrictor effects in arterioles closest to capillaries, with declining effects in descending branch orders approaching feed arterioles. Using our theoretical flow model, simulations assessed total flow and red blood cell (RBC) flow heterogeneity within the network using our experimental pharmacological data. Using our experimental inputs, we are the first to illustrate that spatial differences in ligand sensitivity enable the SNS to modify bulk tissue blood flow and RBC distribution differentially in branching networks.Grant Funding Source: NSERC

Analysis of growth factor signaling in genetically diverse breast cancer lines

BackgroundSoluble growth factors present in the microenvironment play a major role in tumor development, invasion, metastasis, and responsiveness to targeted therapies. While the biochemistry of growth factor-dependent signal transduction has been studied extensively in individual cell types, relatively little systematic data are available across genetically diverse cell lines.ResultsWe describe a quantitative and comparative dataset focused on immediate-early signaling that regulates the AKT (AKT1/2/3) and ERK (MAPK1/3) pathways in a canonical panel of well-characterized breast cancer lines. We also provide interactive web-based tools to facilitate follow-on analysis of the data. Our findings show that breast cancers are diverse with respect to ligand sensitivity and signaling biochemistry. Surprisingly, triple negative breast cancers (TNBCs; which express low levels of ErbB2, progesterone and estrogen receptors) are the most broadly responsive to growth factors and HER2amp cancers (which overexpress ErbB2) the least. The ratio of ERK to AKT activation varies with ligand and subtype, with a systematic bias in favor of ERK in hormone receptor positive (HR+) cells. The factors that correlate with growth factor responsiveness depend on whether fold-change or absolute activity is considered the key biological variable, and they differ between ERK and AKT pathways.ConclusionsResponses to growth factors are highly diverse across breast cancer cell lines, even within the same subtype. A simple four-part heuristic suggests that diversity arises from variation in receptor abundance, an ERK/AKT bias that depends on ligand identity, a set of factors common to all receptors that varies in abundance or activity with cell line, and an “indirect negative regulation” by ErbB2. This analysis sets the stage for the development of a mechanistic and predictive model of growth factor signaling in diverse cancer lines. Interactive tools for looking up these results and downloading raw data are available at http://lincs.hms.harvard.edu/niepel-bmcbiol-2014/.

Lysine Methylation of Progesterone Receptor at Activation Function 1 Regulates both Ligand-independent Activity and Ligand Sensitivity of the Receptor

Progesterone receptor (PR) exists in two isoforms, PRA and PRB, and both contain activation functions AF-1 and AF-2. It is believed that AF-1 is primarily responsible for the ligand-independent activity, whereas AF-2 mediates ligand-dependent PR activation. Although more than a dozen post-translational modifications of PR have been reported, no post-translational modification on AF-1 or AF-2 has been reported. Using LC-MS/MS-based proteomic analysis, this study revealed AF-1 monomethylation at Lys-464. Mutational analysis revealed the remarkable importance of Lys-464 in regulating PR activity. Single point mutation K464Q or K464A led to ligand-independent PR gel upshift similar to the ligand-induced gel upshift. This upshift was associated with increases in both ligand-dependent and ligand-independent PR phosphorylation and PR activity due to the hyperactivation of AF-1. In contrast, mutation of Lys-464 to the bulkier phenylalanine to mimic the effect of methylation caused a drastic decrease in PR activity. Importantly, PR-K464Q also showed heightened ligand sensitivity, and this was associated with increases in its functional interaction with transcription co-regulators NCoR1 and SRC-1. These results suggest that monomethylation of PR at Lys-464 probably has a repressive effect on AF-1 activity and ligand sensitivity.

Antipsychotic agent thioridazine sensitizes renal carcinoma Caki cells to TRAIL-induced apoptosis through reactive oxygen species-mediated inhibition of Akt signaling and downregulation of Mcl-1 and c-FLIP(L)

Thioridazine has been known as an antipsychotic agent, but it also has anticancer activity. However, the effect of thioridazine on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitization has not yet been studied. Here, we investigated the ability of thioridazine to sensitize TRAIL-mediated apoptosis. Combined treatment with thioridazine and TRAIL markedly induced apoptosis in various human carcinoma cells, including renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MDA-MB231), and glioma (U251MG) cells, but not in normal mouse kidney cells (TMCK-1) and human normal mesangial cells. We found that thioridazine downregulated c-FLIP(L) and Mcl-1 expression at the post-translational level via an increase in proteasome activity. The overexpression of c-FLIP(L) and Mcl-1 overcame thioridazine plus TRAIL-induced apoptosis. We further observed that thioridazine inhibited the Akt signaling pathway. In contrast, although other phosphatidylinositol-3-kinase/Akt inhibitors (LY294002 and wortmannin) sensitized TRAIL-mediated apoptosis, c-FLIP(L) and Mcl-1 expressions were not altered. Furthermore, thioridazine increased the production of reactive oxygen species (ROS) in Caki cells, and ROS scavengers (N-acetylcysteine, glutathione ethyl ester, and trolox) inhibited thioridazine plus TRAIL-induced apoptosis, as well as Akt inhibition and the downregulation of c-FLIP(L) and Mcl-1. Collectively, our study demonstrates that thioridazine enhances TRAIL-mediated apoptosis via the ROS-mediated inhibition of Akt signaling and the downregulation of c-FLIP(L) and Mcl-1 at the post-translational level.

2P112 新世界ザルの苦味受容体TAS2R1およびTAS2R4のリガンド感受性の種間差と進化(03. 膜蛋白質,ポスター,第52回日本生物物理学会年会(2014年度))

2P112 新世界ザルの苦味受容体TAS2R1およびTAS2R4のリガンド感受性の種間差と進化(03. 膜蛋白質,ポスター,第52回日本生物物理学会年会(2014年度))

Modulation of Chicken ASIC1 by 2-Guanidine-4-Methylquinazoline (GMQ) in the Absence and Presence of Psalmotoxin-1

Acid-sensing ion channels (ASICs) are trimeric, sodium-selective channels that sense changes in extracellular acidity and are part of the epithelium sodium channel/degenerin (ENaC/DEG) family of ion channels. ASICs are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds, such as amiloride and 2-guanidine-4-methylquinazoline (GMQ). The nonproton ligand GMQ has been shown to stimulate ASIC3 by expanding the pH range of the ASIC window current, but decreased the sensitivity of other ASIC subtypes to protons. The effect of GMQ on chicken ASIC1 (cASIC1), which has been used to elucidate the protein crystal structures, is unknown. Furthermore, cASIC1 exhibits unique channel gating properties, including the spider toxin ASIC1a Psalmotoxin-1 (PcTx1) induced activation. We sought to elucidate the interaction of GMQ, PcTx1, and cASIC1 using whole-cell and outside-out patch clamp electrophysiology to provide additional insight into the nonproton ligand interaction with a structurally characterized ASIC construct. Our studies revealed GMQ increases the cASIC1 proton sensitivity, as observed by a leftward shift in the proton activation curve. When alone, the nonproton ligand failed to activate cASIC1. Additionally, we observed GMQ concentration-dependent enhancement of the cASIC1 PcTx1 persistent current. Our data suggests that GMQ may have multiple sites of action on cASIC1 and may act as a molecular wedge that forces the desensitized ASIC into an open state. We anticipate that the revelation of GMQ stimulation in the cASIC1 subtype will warrant further investigations into nonproton ligand sensitivity in other ASIC subtypes and provide the foundation for the design of novel ligands that exploit the nonproton ligand site to influence ASIC activity.

Novel Piperazine-based Compounds Inhibit Microtubule Dynamics and Sensitize Colon Cancer Cells to Tumor Necrosis Factor-induced Apoptosis

We recently identified a series of mitotically acting piperazine-based compounds that potently increase the sensitivity of colon cancer cells to apoptotic ligands. Here we describe a structure-activity relationship study on this compound class and identify a highly active derivative ((4-(3-chlorophenyl)piperazin-1-yl)(2-ethoxyphenyl)methanone), referred to as AK301, the activity of which is governed by the positioning of functional groups on the phenyl and benzoyl rings. AK301 induced mitotic arrest in HT29 human colon cancer cells with an ED50 of ≈115 nm. Although AK301 inhibited growth of normal lung fibroblast cells, mitotic arrest was more pronounced in the colon cancer cells (50% versus 10%). Cells arrested by AK301 showed the formation of multiple microtubule organizing centers with Aurora kinase A and γ-tubulin. Employing in vitro and in vivo assays, tubulin polymerization was found to be slowed (but not abolished) by AK301. In silico molecular docking suggests that AK301 binds to the colchicine-binding domain on β-tubulin, but in a novel orientation. Cells arrested by AK301 expressed elevated levels of TNFR1 on their surface and more readily activated caspases-8, -9, and -3 in the presence of TNF. Relative to other microtubule destabilizers, AK301 was the most active TNF-sensitizing agent and also stimulated Fas- and TRAIL-induced apoptosis. In summary, we report a new class of mitosis-targeting agents that effectively sensitizes cancer cells to apoptotic ligands. These compounds should help illuminate the role of microtubules in regulating apoptotic ligand sensitivity and may ultimately be useful for developing agents that augment the anti-cancer activities of the immune response.

Functional Cooperation of Metabotropic Adenosine and Glutamate Receptors Regulates Postsynaptic Plasticity in the Cerebellum

G-protein-coupled receptors (GPCRs) may form heteromeric complexes and cooperatively mediate cellular responses. Although heteromeric GPCR complexes are suggested to occur in many neurons, their contribution to neuronal function remains unclear. We address this question using two GPCRs expressed in cerebellar Purkinje cells: adenosine A1 receptor (A1R), which regulates neurotransmitter release and neuronal excitability in central neurons, and type-1 metabotropic glutamate receptor (mGluR1), which mediates cerebellar long-term depression, a form of synaptic plasticity crucial for cerebellar motor learning. We examined interaction between these GPCRs by immunocytochemical, biochemical, and Förster resonance energy transfer analyses in cultured mouse Purkinje cells and heterologous expression cells. These analyses revealed that the GPCRs closely colocalized and formed heteromeric complexes on the cell surfaces. Furthermore, our electrophysiological analysis showed that CSF levels (40-400 nm) of adenosine or synthetic A1R agonists with comparable potencies blocked mGluR1-mediated long-term depression of the postsynaptic glutamate-responsiveness (glu-LTD) of cultured Purkinje cells. A similar dose of the A1R agonist decreased the ligand affinity of mGluR1 and did not affect depolarization-induced Ca(2+) influx, which is an essential factor in inducing glu-LTD. The A1R agonist did not affect glu-LTD mimicked by direct activation of protein kinase C. These results suggest that A1R blocked glu-LTD by decreasing the ligand sensitivity of mGluR1, but not the coupling efficacy from mGluR1 to the intracellular signaling cascades. These findings provide a new insight into neuronal GPCR signaling and demonstrate a novel regulatory mechanism of synaptic plasticity.

Two Sets of Metal Organic Frameworks along the Lanthanide Series Constructed by 2,3-Dimethylsuccinate: Structures, Topologies, and Strong Emission without Ligand Sensitization

Reactions in aqueous solution under hydrothermal conditions between (±)-2,3-dimethylsuccinic acid and lanthanide chlorides lead to two different isostructural types with chemical formulas [Ln2(C6H8O4)3(H2O)2] Ln(III) = Pr–Eu (except Pm) (Type I, compounds 1–4) and [Ln2(C6H8O4)3] Ln(III) = Tb–Yb (except Tm) (Type II, compounds 5–9). The crystal structure has been solved for the Pr (1)-, Sm (3)-, and Ho (7)-containing compounds by means of single-crystal XRD methods, whereas powder XRD Rietveld refinement was used for the rest of the MOFs. Compounds 1–4 crystallize in the triclinic space group P1̅, whereas compounds 5–9 belong to the tetragonal space group P43212. Type I and II compounds are 3D frameworks consisting of chains of [LnO8(H2O)] or [LnO8] polyhedra, respectively, linked by dimethylsuccinate anions, giving rise to I1O2 connectivity. All the compounds were characterized by X-ray diffraction, variable-temperature Fourier transform infrared spectroscopy, and thermal analysis. An exhaustive topological study was performed in comparison with other related compounds. The photoluminescent (PL) properties for compounds 3, 4, and 5 have been also explored, indicating that a metal-centered luminescent process takes place.

Cyclic Nucleotide-Gated Channel Subunit Glycosylation Regulates Matrix Metalloproteinase-Dependent Changes in Channel Gating

Cyclic-nucleotide gated (CNG) channels are essential for phototransduction within retinal photoreceptors. We have demonstrated previously that the enzymatic activity of matrix metalloproteinase-2 and -9, members of the matrix metalloproteinase (MMP) family of extracellular, Ca(2+)- and Zn(2+)-dependent proteases, enhances the ligand sensitivity of both rod (CNGA1 and CNGB1) and cone (CNGA3 and CNGB3) CNG channels. Additionally, we have observed a decrease in the maximal CNG channel current (Imax) that begins late during MMP-directed gating changes. Here we demonstrate that CNG channels become nonconductive after prolonged MMP exposure. Concurrent with the loss of conductive channels is the increased relative contribution of channels exhibiting nonmodified gating properties, suggesting the presence of a subpopulation of channels that are protected from MMP-induced gating effects. CNGA subunits are known to possess one extracellular core glycosylation site, located at one of two possible positions within the turret loop near the pore-forming region. Our results indicate that CNGA glycosylation can impede MMP-dependent modification of CNG channels. Furthermore, the relative position of the glycosylation site within the pore turret influences the extent of MMP-dependent proteolysis. Glycosylation at the site found in CNGA3 subunits was found to be protective, while glycosylation at the bovine CNGA1 site was not. Relocating the glycosylation site in CNGA1 to the position found in CNGA3 recapitulated CNGA3-like protection from MMP-dependent processing. Taken together, these data indicate that CNGA glycosylation may protect CNG channels from MMP-dependent proteolysis, consistent with MMP modification of channel function having a requirement for physical access to the extracellular face of the channel.

EML1 (CNG-Modulin) Controls Light Sensitivity in Darkness and under Continuous Illumination in Zebrafish Retinal Cone Photoreceptors

The ligand sensitivity of cGMP-gated (CNG) ion channels in cone photoreceptors is modulated by CNG-modulin, a Ca(2+)-binding protein. We investigated the functional role of CNG-modulin in phototransduction in vivo in morpholino-mediated gene knockdown zebrafish. Through comparative genomic analysis, we identified the orthologue gene of CNG-modulin in zebrafish, eml1, an ancient gene present in the genome of all vertebrates sequenced to date. We compare the photoresponses of wild-type cones with those of cones that do not express the EML1 protein. In the absence of EML1, dark-adapted cones are ∼5.3-fold more light sensitive than wild-type cones. Previous qualitative studies in several nonmammalian species have shown that immediately after the onset of continuous illumination, cones are less light sensitive than in darkness, but sensitivity then recovers over the following 15-20 s. We characterize light sensitivity recovery in continuously illuminated wild-type zebrafish cones and demonstrate that sensitivity recovery does not occur in the absence of EML1.

Induction of Mesenchymal Stem Cell Chondrogenesis Through Sequential Administration of Growth Factors Within Specific Temporal Windows

Human mesenchymal stem cells (hMSCs) are capable of differentiating into chondrocyte-like cells but fail to produce the quality or quantity of cartilage matrix compared to articular chondrocytes using current differentiation protocols. In this study, we aim to improve the chondrogenic differentiation of hMSCs through the sequential administration of multiple growth factors (GFs). We began by looking at differentiating hMSCs' cell surface GF receptor expression every 3 days throughout differentiation using flow cytometry and found that not only was receptor expression dynamic throughout differentiation, but ligand sensitivity was positively correlated with receptor expression, suggesting that differentiating hMSCs may have varying GF requirements depending on their stage of differentiation. We then constructed GF sequences by administering several prochondrogenic GFs singly every 3 days throughout differentiation and assaying the expression of a variety of cartilage-related genes using qPCR. The resulting chondrocytic phenotype of sequentially induced hMSCs was then compared to that of hMSCs induced under standard culture conditions using qPCR, dimethylmethylene blue assay, and histology. We found that while the initial GF sequence was unable to improve hMSC chondrogenesis, withdrawal of GF treatment at Day 9 of differentiation in pellet culture vastly improved the success of differentiation beyond that induced by TGFβ1 alone. Additional modifications allowed us to further improve chondrogenesis to levels comparable to that obtained by co-administration of TGFβ1 and BMP7 throughout differentiation. Taken together, we demonstrated the ability to improve the chondrocytic phenotype of differentiated hMSCs through the sequential administration of multiple GFs.

Functional Evolution of a Multigene Family: Orthologous and Paralogous Pheromone Receptor Genes in the Turnip Moth, Agrotis segetum

Lepidopteran pheromone receptors (PRs), for which orthologies are evident among closely related species, provide an intriguing example of gene family evolution in terms of how new functions may arise. However, only a limited number of PRs have been functionally characterized so far and thus evolutionary scenarios suffer from elements of speculation. In this study we investigated the turnip moth Agrotis segetum, in which female moths produce a mixture of chemically related pheromone components that elicit specific responses from receptor cells on male antennae. We cloned nine A. segetum PR genes and the Orco gene by degenerate primer based RT-PCR. The nine PR genes, named as AsegOR1 and AsegOR3-10, fall into four distinct orthologous clusters of known lepidopteran PRs, of which one contains six paralogues. The paralogues are under relaxed selective pressure, contrasting with the purifying selection on other clusters. We identified the receptors AsegOR9, AsegOR4 and AsegOR5, specific for the respective homologous pheromone components (Z)-5-decenyl, (Z)-7-dodecenyl and (Z)-9-tetradecenyl acetates, by two-electrode voltage clamp recording from Xenopus laevis oocytes co-expressing Orco and each PR candidate. These receptors occur in three different orthologous clusters. We also found that the six paralogues with high sequence similarity vary dramatically in ligand selectivity and sensitivity. Different from AsegOR9, AsegOR6 showed a relatively large response to the behavioural antagonist (Z)-5-decenol, and a small response to (Z)-5-decenyl acetate. AsegOR1 was broadly tuned, but most responsive to (Z)-5-decenyl acetate, (Z)-7-dodecenyl acetate and the behavioural antagonist (Z)-8-dodecenyl acetate. AsegOR8 and AsegOR7, which differ from AsegOR6 and AsegOR1 by 7 and 10 aa respectively, showed much lower sensitivities. AsegOR10 showed only small responses to all the tested compounds. These results suggest that new receptors arise through gene duplication, and relaxed evolutionary constraints or positive selection among paralogues allow functional divergence to occur in spite of purifying selection being the norm.

Abstract C14: Modeling the dynamics of caspase 8 activity after TRAIL stimulation reveals DISC features controlling cell fate in vitro

Abstract Ligands and DR4/5-receptor agonist antibodies such as TRAIL or Apomab trigger apoptosis in tumor cells and are currently being tested in clinical trials. Exposure of cells to these ligands induces DISC (Death receptor Induced Signaling Complex) formation. When formed, these complexes activate caspase 8 which in turn cleaves Bid and triggers mitochondrial outer membrane permeabilization (MOMP) leading to apoptosis. Although promising, drugs targeting this pathway have stalled in Phase II/III of clinical trials because of variable efficacy. Many mechanisms of resistance have been proposed but no quantitative model has been built to evaluate and compare these different hypotheses. In this study, we aim to understand which features of DISC dynamics control cell fate when exposed to DR ligands, and how genomic variations or pharmaceutical drugs affect these features and ultimately ligand sensitivity. For this task, we will build a quantitative and mechanistic model of DISC activation valid in different genetic backgrounds. Using live cell imaging and a FRET reporter of Bid cleavage, we monitored caspase 8 activity at single cell level after exposure to TRAIL. In addition, we used a MOMP reporter to determine the cells undergoing apoptosis. To identify differences between cells that survive or die, we used doses of ligands that kill only a fraction of the cell population. Using a phenomenological model, we derived parameters that characterize DISC activation based on the dynamics of the FRET reporter. These parameters are significantly different between the cells that undergo MOMP and the ones that survive. We then determined how these parameters change when using different ligands (Apomab with or without clustering agent) or in cells with different genetic backgrounds (FLIP-L/S or BCL-2 overexpression). These results define quantitative relationships between these DISC parameters, key proteins of the apoptotic pathways and cell fate. Based on our knowledge of DISC formation and caspase 8 activation, we built a detailed mechanistic model of the apoptotic pathway using PySB, a framework for rule-based modeling. We calibrated the model parameters to fit the sensitivity of DISC activation to ligand concentration and FLIP expression level. This model allowed us to predict how DISC dynamics can be pharmacologically modulated to impact cell fate, and how pharmacological perturbations of other modules of the apoptotic pathway, such as the mitochondria and effector caspases, can sensitize cells to a given ligand stimulation. In particular, our model predicts which drug combinations are effective in different genetic backgrounds, a prediction that we tested experimentally. Citation Format: Jeremie Roux, Marc Hafner, Samuel Bandara, Joshua Sims, Peter K. Sorger. Modeling the dynamics of caspase 8 activity after TRAIL stimulation reveals DISC features controlling cell fate in vitro. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C14.

Expressomal approach for comprehensive analysis and visualization of ligand sensitivities of xenoestrogen responsive genes

Although biological effects of endocrine disrupting chemicals (EDCs) are often observed at unexpectedly low doses with occasional nonmonotonic dose-response characteristics, transcriptome-wide profiles of sensitivities or dose-dependent behaviors of the EDC responsive genes have remained unexplored. Here, we describe expressome analysis for the comprehensive examination of dose-dependent gene responses and its applications to characterize estrogen responsive genes in MCF-7 cells. Transcriptomes of MCF-7 cells exposed to varying concentrations of representative natural and xenobiotic estrogens for 48 h were determined by microarray and used for computational calculation of interpolated approximations of estimated transcriptomes for 300 doses uniformly distributed in log space for each chemical. The entire collection of these estimated transcriptomes, designated as the expressome, has provided unique opportunities to profile chemical-specific distributions of ligand sensitivities for large numbers of estrogen responsive genes, revealing that at low concentrations estrogens generally tended to suppress rather than to activate transcription. Gene ontology analysis demonstrated distinct functional enrichment between high- and low-sensitivity estrogen responsive genes, supporting the notion that a single EDC chemical can cause qualitatively distinct biological responses at different doses. Expressomal heatmap visualization of dose-dependent induction of Bisphenol A inducible genes showed a weak gene activation peak at a very low concentration range (ca. 0.1 nM) in addition to the main, strong gene activation peak at and above 100 nM. Thus, expressome analysis is a powerful approach to understanding the EDC dose-dependent dynamic changes in gene expression at the transcriptomal level, providing important information on the overall profiles of ligand sensitivities and nonmonotonic responses.

Extracting Modulation Transfer Function of the Visual System from Contrast Sensitivity Function in External Noise

To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells. Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3',5'-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes. Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium. These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels.

Developmental control of the melanocortin-4 receptor by MRAP2 proteins in zebrafish.

The melanocortin-4 receptor (MC4R) is essential for control of energy homeostasis in vertebrates. MC4R interacts with melanocortin receptor accessory protein 2 (MRAP2) in vitro, but its functions in vivo are unknown. We found that MRAP2a, a larval form, stimulates growth of zebrafish by specifically blocking the action of MC4R. In cell culture, this protein binds MC4R and reduces the ability of the receptor to bind its ligand, α-melanocyte-stimulating hormone (α-MSH). A paralog, MRAP2b, expressed later in development, also binds MC4R but increases ligand sensitivity. Thus, MRAP2 proteins allow for developmental control of MC4R activity, with MRAP2a blocking its function and stimulating growth during larval development, whereas MRAP2b enhances responsiveness to α-MSH once the zebrafish begins feeding, thus increasing the capacity for regulated feeding and growth.

Divergent Sequence Tunes Ligand Sensitivity in Phospholipid-regulated Hormone Receptors

The members of the NR5A subfamily of nuclear receptors (NRs) are important regulators of pluripotency, lipid and glucose homeostasis, and steroidogenesis. Liver receptor homologue 1 (LRH-1; NR5A2) and steroidogenic factor 1 (SF-1; NR5A1) have therapeutic potential for the treatment of metabolic and neoplastic disease; however, a poor understanding of their ligand regulation has hampered the pursuit of these proteins as pharmaceutical targets. In this study, we dissect how sequence variation among LRH-1 orthologs affects phospholipid (PL) binding and regulation. Both human LRH-1 (hLRH-1) and mouse LRH-1 (mLRH-1) respond to newly discovered medium chain PL agonists to modulate lipid and glucose homeostasis. These PLs activate hLRH-1 by altering receptor dynamics in a newly identified alternate activation function region. Mouse and Drosophila orthologs contain divergent sequences in this region potentially altering PL-driven activation. Structural evidence suggests that these sequence differences in mLRH-1 and Drosophila FTZ-f1 (dmFTZ-f1) confer at least partial ligand independence, making them poor models for hLRH-1 studies; however, the mechanisms of ligand independence remain untested. We show using structural and biochemical methods that the recent evolutionary divergence of the mLRH-1 stabilizes the active conformation in the absence of ligand, yet does not abrogate PL-dependent activation. We also show by mass spectrometry and biochemical assays that FTZ-f1 is incapable of PL binding. This work provides a structural mechanism for the differential tuning of PL sensitivity in NR5A orthologs and supports the use of mice as viable therapeutic models for LRH-1-dependent diseases.

Inhibition of Glycogen Synthase Kinase-3 Increases NKG2D Ligand MICA Expression and Sensitivity to NK Cell–Mediated Cytotoxicity in Multiple Myeloma Cells: Role of STAT3

Engagement of NKG2D and DNAX accessory molecule-1 (DNAM-1) receptors on lymphocytes plays an important role for anticancer response and represents an interesting therapeutic target for pharmacological modulation. In this study, we investigated the effect of inhibitors targeting the glycogen synthase kinase-3 (GSK3) on the expression of NKG2D and DNAM-1 ligands in multiple myeloma (MM) cells. GSK3 is a pleiotropic serine-threonine kinase point of convergence of numerous cell-signaling pathways, able to regulate the proliferation and survival of cancer cells, including MM. We found that inhibition of GSK3 upregulates both MICA protein surface and mRNA expression in MM cells, with little or no effects on the basal expression of the MICB and DNAM-1 ligand poliovirus receptor/CD155. Moreover, exposure to GSK3 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and to enhance the ability of myeloma cells to trigger NK cell-mediated cytotoxicity. We could exclude that increased expression of β-catenin or activation of the heat shock factor-1 (transcription factors inhibited by active GSK3) is involved in the upregulation of MICA expression, by using RNA interference or viral transduction of constitutive active forms. On the contrary, inhibition of GSK3 correlated with a downregulation of STAT3 activation, a negative regulator of MICA transcription. Both Tyr(705) phosphorylation and binding of STAT3 on MICA promoter are reduced by GSK3 inhibitors; in addition, overexpression of a constitutively active form of STAT3 significantly inhibits MICA upregulation. Thus, we provide evidence that regulation of the NKG2D-ligand MICA expression may represent an additional immune-mediated mechanism supporting the antimyeloma activity of GSK3 inhibitors.

Cells surviving fractional killing by TRAIL exhibit transient but sustainable resistance and inflammatory phenotypes

When clonal populations of human cells are exposed to apoptosis-inducing agents, some cells die and others survive. This fractional killing arises not from mutation but from preexisting, stochastic differences in the levels and activities of proteins regulating apoptosis. Here we examine the properties of cells that survive treatment with agonists of two distinct death receptors, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anti-FasR antibodies. We find that "survivor" cells are highly resistant to a second ligand dose applied 1 d later. Resistance is reversible, resetting after several days of culture in the absence of death ligand. "Reset" cells appear identical to drug-naive cells with respect to death ligand sensitivity and gene expression profiles. TRAIL survivors are cross-resistant to activators of FasR and vice versa and exhibit an NF-κB-dependent inflammatory phenotype. Remarkably, reversible resistance is induced in the absence of cell death when caspase inhibitors are present and can be sustained for 1 wk or more, also without cell death, by periodic ligand exposure. Thus stochastic differences in cell state can have sustained consequences for sen-sitivity to prodeath ligands and acquisition of proinflammatory phenotypes. The important role played by periodicity in TRAIL exposure for induction of opposing apoptosis and survival mechanisms has implications for the design of optimal therapeutic agents and protocols.