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1.
Methods Cell Biol ; 132: 319-37, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26928550

RESUMO

The use of biosensors either individually or as part of panels has now become a common technique to capturing signaling events in living cells. Such biosensors have become particularly important for probing biased signaling and allostery in G protein-coupled receptor drug screening efforts. However, assumptions about the portability of such biosensors between cell types may lead to misinterpretation of drug effects on specific signaling pathways in a given cellular context. Further, the output of a particular biosensor may be different depending on where it is localized in a cell. Here, we discuss strategies to mitigate these concerns which should feed into future biosensor design and usage.


Assuntos
Técnicas Biossensoriais , Transdução de Sinais , Núcleo Celular/enzimologia , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases , Receptores Acoplados a Proteínas-G/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
2.
J Org Chem ; 78(4): 1547-52, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23351142

RESUMO

The palladium-catalyzed alkoxycarbonylation of an α-chloro ketone can be efficiently combined to a Michael addition reaction in a new two-step domino reaction, allowing the synthesis of original highly functionalized α-alkylated ß-ketoesters. The scope of the reaction was extended to several α-chloro ketones and Michael acceptors with moderate to very good yields.

3.
J Am Chem Soc ; 134(29): 12281-8, 2012 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-22713106

RESUMO

Treatment of alkenes such as 3-hexene, 3-octene, and 1-cyclohexyl-1-butene with the N-heterocyclic carbene (NHC)-derived borane 2 and catalytic HNTf(2) (Tf = trifluoromethanesulfonyl (CF(3)SO(2))) effects hydroboration at room temperature. With 3-hexene, surprisingly facile migration of the boron atom from C(3) of the hexyl group to C(2) was observed over a time scale of minutes to hours. Oxidative workup gave a mixture of alcohols containing 2-hexanol as the major product. A similar preference for the C(2) alcohol was observed after oxidative workup of the 3-octene and 1-cyclohexyl-1-butene hydroborations. NHC-borenium cations (or functional equivalents) are postulated as the species that accomplish the hydroborations, and the C(2) selective migrations are attributed to the four-center interconversion of borenium cations with cationic NHC-borane-olefin π-complexes.


Assuntos
Alcenos/química , Boranos/química , Boro/química , Metano/análogos & derivados , Catálise , Íons/química , Metano/química
4.
Org Biomol Chem ; 9(13): 4714-24, 2011 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-21537514

RESUMO

Dioxazaborocanes are boronic adducts obtained by condensation of diethanolamine derivatives with boronic compounds. They were first described in the mid-1950's as a practical way to isolate a boronic adduct. Their use has for a long time been restricted to this purpose for the isolation and characterisation of either a final product or a boronic intermediate. Only recently have they been directly involved in chemical transformations in which they proved equivalent or superior to their acid counterpart. In the meantime they have also been used as protected boronic acids. We wish to show in this report that they will likely represent a fluoride-free alternative to organotrifluoroborate salts and therefore an area of intense development.

5.
Biophys J ; 99(12): 4037-46, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21156147

RESUMO

Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/metabolismo , Cor , AMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Receptores Acoplados a Proteínas-G/metabolismo
6.
Chem Commun (Camb) ; 46(15): 2677-9, 2010 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-20449335

RESUMO

Boronic esters have long been considered as poor partners in cross-coupling reactions with arene diazoniums. Here is reported an unprecedented application of self-activated boronic esters in a base-free cross-coupling reaction with diazonium salts under mild and user friendly conditions.

7.
Mol Pharmacol ; 74(1): 162-72, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18403719

RESUMO

In recent years, several studies have demonstrated that different ligands can have distinct efficacy profiles toward various signaling pathways through a unique receptor. For example, beta1-adrenergic compounds that are inverse agonists toward the adenylyl cyclase (AC) can display agonist activity for the mitogen-activated protein kinase (MAPK) pathway. Such a phenomenon, often termed functional selectivity, has now been clearly established for many G protein-coupled receptors when considering distinct signaling output. However, the possibility that ligands could selectively engage distinct effectors to activate a single signaling output by promoting specific receptor conformations has not been extensively examined. Here, we took advantage of the fact that isoproterenol, bucindolol and propranolol (full, partial, and inverse agonists for the AC pathway, respectively) all activate MAPK through the beta1-adrenergic receptor (beta1AR) to probe such conformational-biased signaling. Although the three compounds stimulated MAPK in a src-dependent manner, isoproterenol acted through both Galpha(i)betagamma- and G protein-independent pathways, whereas bucindolol and propranolol promoted MAPK activation through the G protein-independent pathway only. The existence of such distinct signaling cascades linking beta1AR to MAPK activation was correlated with ligand-specific conformational rearrangements of receptor/G protein complexes measured by bioluminescence resonance energy transfer. Taken together, our data indicate that discrete local conformational changes can selectively promote the recruitment of distinct proximal signaling partners that can engage distinct signaling outputs and/or converge on the same signaling output.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Conformação Proteica , Receptores Adrenérgicos beta 1/metabolismo , Transdução de Sinais , Linhagem Celular , AMP Cíclico/biossíntese , Hemaglutininas/metabolismo , Humanos , Rim/citologia , Ligantes , Fosforilação , Receptores Adrenérgicos beta 1/química , Transfecção
8.
Cell Signal ; 19(1): 32-41, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16857342

RESUMO

The V2 vasopressin receptor (V2R) activates the mitogen activated protein kinases (MAPK) ERK1/2 through a mechanism involving the scaffolding protein beta arrestin. Here we report that this activating pathway is independent of G alpha s, G alpha i, G alpha q or G betagamma and that the V2R-mediated activation of G alpha s inhibits ERK1/2 activity in a cAMP/PKA-dependent manner. In the HEK293 cells studied, the beta arrestin-promoted activation was found to dominate over the PKA-mediated inhibition of the pathway, leading to a strong vasopressin-stimulated ERK1/2 activation. Despite the strong MAPK activation and in contrast with other GPCR, V2R did not induce any significant increase in DNA synthesis, consistent with the notion that the stable interaction between V2R and beta arrestin prevents signal propagation to the nucleus. Beta arrestin was found to be essential for the ERK1/2 activation, indicating that the recruitment of the scaffolding protein is necessary and sufficient to initiate the signal in the absence of any other stimulatory cues. Based on the use of selective pharmacological inhibitors, dominant negative mutants and siRNA, we conclude that the beta arrestin-dependent activation of ERK1/2 by the V2R involves c-Src and a metalloproteinase-dependent trans-activation event. These findings demonstrate that beta arrestin is a genuine signalling initiator that can, on its own, engage a MAPK activation machinery upon stimulation of a GPCR by its natural ligand.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Receptores de Vasopressinas/fisiologia , Animais , Arrestinas/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Humanos , Metaloproteinases da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Ativação Transcricional , beta-Arrestinas , Quinases da Família src
9.
EMBO J ; 25(12): 2698-709, 2006 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-16724110

RESUMO

The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT-Pep-27) did not affect basal signaling activity but almost completely abolished agonist-promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT-Pep-27 prevented both agonist-promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.


Assuntos
Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Proteína Quinase C/metabolismo , Receptores de GABA-B/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Dimerização , Proteínas de Ligação ao GTP/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Sensíveis a N-Etilmaleimida/antagonistas & inibidores , Neurônios/citologia , Peptídeos/química , Fosforilação , Ligação Proteica , Subunidades Proteicas , Transporte Proteico , Ratos , Termodinâmica
10.
Biol Chem ; 384(1): 117-23, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12674505

RESUMO

G Protein-coupled receptor dimerization/oligomerization has been well established during the last several years. Studies have demonstrated the existence of dimers/digomers both in vitro and in living cells. However, a thorough characterization of the biochemical nature of receptor dimers and oligomers as well as their occurrence at the cell surface has not been properly addressed. In this study, we show that both beta2-adrenergic receptor (beta2AR) dimers and oligomers exist at the plasma membrane and that the detection of such species, following receptor solubilization and resolution by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), does not result from the formation of spurious disulfide bonds during cell lysis. Moreover, our results indicate that the biochemical nature of beta2AR dimers is different from that of the oligomers. Although both complexes are partially resistant to SDS denaturation, disulfide bonding is absolutely required for the stability of beta2AR oligomers but not dimers in SDS-PAGE. Indeed, dimeric species can be detected even in the presence of high concentrations of reducing and alkylating agents. Although the different biochemical nature of the dimers and oligomers may be indicative of distinct biological roles in cells, additional studies will be required to further elucidate the biosynthesis and function of these receptor forms.


Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Western Blotting , Linhagem Celular , Cromatografia de Afinidade , Reagentes para Ligações Cruzadas , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/metabolismo , Humanos , Imunoquímica , Membranas/metabolismo , Receptores Adrenérgicos beta 2/química , Ultrafiltração
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