Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis

Atwood, Brady K.; Lopez, Jacqueline; Wager‐Miller, James; Mackie, Ken; Straiker, Alex

doi:10.1186/1471-2164-12-14

Cited by 343 publications

(338 citation statements)

References 71 publications

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“…This approach provides relatively rapid real-time response, and assays of [Ca] i concentration can be amenable to studying receptor desensitization. 34,35 Interestingly, we were unable to activate intracellular Ca release in AtT-20 cells via MOR or any of the Gα q receptors reportedly expressed in this cell line 36 (data not shown).…”

Section: Discussionmentioning

confidence: 92%

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Knapman

Santiago

et al. 2013

SLAS Discovery

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Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary "no-wash" fluorescent membrane potential dye to act as a reporter of µ-opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.

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Section: Discussionmentioning

confidence: 92%

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Knapman

Santiago

et al. 2013

SLAS Discovery

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“…To test whether a specific PLC isozyme is involved in TRPC4 activation, we knocked down PLC isoforms typically associated with G q/11 and Ca 2+ signaling in HEK293 cells (35) by siRNA. Intriguingly, knockdown of PLCδ1 but not PLCδ3, PLCβ1, or PLCβ3 inhibited TRPC4 activation (SI Appendix, Fig.…”

Section: Trpc4 Activation Requires Coincident G I/o Stimulation and Plcmentioning

confidence: 99%

Critical roles of G _i/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels

Thakur

Tian

Jeon

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP 2 ), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of G i/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCβ pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and G i/o -coupled μ opioid receptor, μ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca 2+ and PIP 2 . Reducing PIP 2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak G i/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of G i/o -mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident G i/o , Ca 2+ , and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.Canonical TRPs (TRPC1-7) are the most homologous to the prototypical Drosophila TRP and are believed to be activated downstream of phospholipase C (PLC) (1). In both heterologous and native systems, stimulating PLCβ via the G q/11 subgroup of G proteins is commonly used to activate TRPC channels. Recent studies, however, also suggest a role for G i/o subgroup of G proteins in the activation of TRPC4/C5 (2-4).TRPC4 is implicated in the regulation of microvascular permeability (5), renal cancer proliferation (6, 7), neurotransmitter release (8), intestinal contraction and motility (9), neurite extension (10), epileptiform burst firing, and seizure-induced neurodegeneration (11). The channel mediates Na + and Ca 2+ influx, causing membrane depolarization and intracellular Ca 2+ concentration ([Ca 2+ ] i ) elevation, which in turn alter cell function (12). Although advances have been made in demonstrating TRPC4 channel activation under G i/o and/or PLC stimulation, as well as its dependence on [Ca 2+ ] i , a precise description of signaling events underlying the mechanism of TRPC4 activation remains elusive.Here, we distinguished the contributions of G q/11 and G i/o pathways to TRPC4 activation and uncovered a strict codependence on G i/o and PLC pathways. We focuse...

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“…56 To verify the subtype of Ga i proteins, first, we tested the effect of the subtype of wild-type (WT) Ga i proteins on mTRPC4b currents. WT Ga i2 or Ga i3 protein itself increased the mTRPC4b currents in HEK293 cells without activator GTPgS.…”

Section: Trpc4 Channelsmentioning

confidence: 99%

The Roles of Rasd1 small G proteins and leptin in the activation of TRPC4 transient receptor potential channels

et al. 2015

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#These authors contributed equally to this work TRPC4 is important regulators of electrical excitability in gastrointestinal myocytes, pancreatic b-cells and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Ga i/o and Ga q protein coupled receptor or epidermal growth factor and leptin in particular. However, our understanding of the roles of small G proteins and leptin on TRPC4 channels is still rudimentary. We discuss potential roles for Rasd1 small G protein and leptin in channel activation in addition to their known role in cellular signaling.

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Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis

Cited by 343 publications

References 71 publications

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Critical roles of G _i/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels

The Roles of Rasd1 small G proteins and leptin in the activation of TRPC4 transient receptor potential channels

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Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis

Cited by 343 publications

References 71 publications

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Critical roles of G i/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels

The Roles of Rasd1 small G proteins and leptin in the activation of TRPC4 transient receptor potential channels

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Critical roles of G _i/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels