Europium-labeled GTP as a general nonradioactive substitute for [35S]GTPγS in high-throughput G protein studies

Koval, Alexey; Kopein, Damir; Purvanov, Vladimir; Katanaev, Vladimir L.

doi:10.1016/j.ab.2009.10.028

Cited by 26 publications

(27 citation statements)

References 31 publications

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“…The resultant plasmid pQE32-CG9139, together with pQE32-Rab5 (76) and pQE32-Ga o (77), was used for the bacterial expression and purification of N-terminally His 6 -tagged proteins, as described previously (76). The coding sequences of fz and fz2 lacking the predicted signal sequences (Swiss-Prot) were cloned C-terminally to the MBP sequence in the plasmid pMALpoly for plasma membrane-directed bacterial expression, as described previously (43).…”

Section: Cloning and Protein Expressionmentioning

confidence: 99%

A Direct and Functional Interaction Between G _o and Rab5 During G Protein–Coupled Receptor Signaling

2010

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Rab5 is a small guanosine triphosphatase (GTPase) that regulates the early stages of endocytosis and is conserved in eukaryotes. Rab5 regulates the internalization of receptors and other membrane-associated signaling proteins. The function of Rab5 in these processes is considered relatively passive, so that the endocytic capacity of Rab5 is used during, for example, beta-arrestin-dependent internalization of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs). Direct recruitment or activation of Rab5 by the components of these signaling pathways has not been reported. Here, we demonstrate an interaction of Drosophila Rab5 and an immediate transducer of GPCR signaling, the G protein G(o), in vitro and in vivo. Rab5 and G(o) bound to each other as purified proteins, as well as in fly extracts. In cellular assays, G(o) led to Rab5 activation and endosome fusion. We further showed that the G(o)-Rab5 interaction functioned in Drosophila planar cell polarity and Wingless signal transduction, pathways initiated by GPCRs of the Frizzled (Fz) family. Additionally, the recycling Rab GTPases Rab4 and Rab11 functioned in Fz- and G(o)-mediated signaling to favor planar cell polarity over canonical Wingless signaling. The interplay between heterotrimeric G proteins and Rab GTPases controlled receptor internalization, revealing a previously uncharacterized regulatory mechanism in GPCR signaling.

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Section: Cloning and Protein Expressionmentioning

confidence: 99%

A Direct and Functional Interaction Between G _o and Rab5 During G Protein–Coupled Receptor Signaling

2010

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“…Another highly specific way to monitor substances that affect FZDs would be to observe receptor internalization (Chen et al, 2009b) or the recruitment of ␤-arrestin (Verkaar et al, 2008) in living cells. Furthermore, monitoring second messenger production or G protein activation could be suitable and well established methods for drug screening (Koval et al, 2010). Dirnberger and Seuwen (2007) , 2006).…”

Section: Frizzled-targeting Drugsmentioning

confidence: 99%

International Union of Basic and Clinical Pharmacology. LXXX. The Class Frizzled Receptors

Schulte¹

2010

Pharmacol Rev

195

231

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“…Recently, Perkin Elmer has developed a non-radioactive time-resolved fluorescence (TRF) GTP-binding assay using lanthanide chelate technology (europium-labeled GTP) [54]. Koval, A., et al, successfully used this Europium-labeled GTP as a general nonradioactive substitute for [ 35 S]GTP S in high throughput G protein studies [55]. But this technology still requires a filtration and washing step, besides, it is far less sensitive than [ 35 S]GTP S binding assays.…”

Section: Gtp Binding Assaysmentioning

confidence: 99%

“…But this technology still requires a filtration and washing step, besides, it is far less sensitive than [ 35 S]GTP S binding assays. Anyhow, GTP S binding assays are still attractive, despite the assay formats are very technically challenging [55].…”

Section: Gtp Binding Assaysmentioning

confidence: 99%

High Throughput Screening (HTS) in Identification New Ligands and Drugable Targets of G Protein-Coupled Receptors (GPCRs)

Wang

Li²,

Zhang³

et al. 2012

CCHTS

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G protein-coupled receptors (GPCRs) which constitute one of the largest and most versatile families of cell surface receptors are involved in a wide spectrum of physiological functions, such as, neuronal transmission, chemotaxis, pacemaker activity and embryonic development. Therefore, in the past a few years GPCR families have become very important targets in pharmaceutical design. However, according to the human genome project, there are approximately 1000 genes encoding GPCRs, only about 200 of GPCRs have known ligands and functions. Searching for ligands of the unknown GPCRs and better modulators of known GPCRs are currently attracting lots of interest. High throughput screening (HTS), which is commonly defined as an automatic process of testing potential drug candidates efficiently, is widely used in drug discovery. In this review, the use of high throughput screening (HTS) in studying GPCRs and the choice of screening technology in different G-protein signaling pathways were summarized.

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Europium-labeled GTP as a general nonradioactive substitute for [35S]GTPγS in high-throughput G protein studies

Cited by 26 publications

References 31 publications

A Direct and Functional Interaction Between G _o and Rab5 During G Protein–Coupled Receptor Signaling

A Direct and Functional Interaction Between G _o and Rab5 During G Protein–Coupled Receptor Signaling

International Union of Basic and Clinical Pharmacology. LXXX. The Class Frizzled Receptors

High Throughput Screening (HTS) in Identification New Ligands and Drugable Targets of G Protein-Coupled Receptors (GPCRs)

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Europium-labeled GTP as a general nonradioactive substitute for [35S]GTPγS in high-throughput G protein studies

Cited by 26 publications

References 31 publications

A Direct and Functional Interaction Between G o and Rab5 During G Protein–Coupled Receptor Signaling

A Direct and Functional Interaction Between G o and Rab5 During G Protein–Coupled Receptor Signaling

International Union of Basic and Clinical Pharmacology. LXXX. The Class Frizzled Receptors

High Throughput Screening (HTS) in Identification New Ligands and Drugable Targets of G Protein-Coupled Receptors (GPCRs)

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A Direct and Functional Interaction Between G _o and Rab5 During G Protein–Coupled Receptor Signaling

A Direct and Functional Interaction Between G _o and Rab5 During G Protein–Coupled Receptor Signaling