Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with β-arrestin 2

Kubale, Valentina; Abramović, Zrinka; Pogačnik, A.; Heding, Anders; Šentjurc, Marjeta; Vrecl, Milka

doi:10.1007/s00441-007-0462-y

Cited by 26 publications

(23 citation statements)

References 59 publications

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“…The ␤Arr translocation to the membrane after activation of N-formyl peptide receptor was not observed in cholesterol-depleted cells (Xue et al, 2004). Our study with OPRM1 and a study with the neurokinin-1 receptor (Kubale et al, 2007) demonstrate that agonist-induced ␤Arr recruitment is substantially attenuated by the disruption of lipid rafts. Moreover, translocation of ␤Arr to lipid raft domains has been observed with the lipid raft-located rhodopsin receptor that does not translocate to nonraft microdomains after light activation (Nair et al, 2002).…”

Section: Discussionmentioning

confidence: 56%

Cholesterol Regulates μ-Opioid Receptor-Induced β-Arrestin 2 Translocation to Membrane Lipid Rafts

Qiu¹,

Wang²,

Law³

et al. 2011

Mol Pharmacol

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-Opioid receptor (OPRM1) is mainly localized in lipid raft microdomains but internalizes through clathrin-dependent pathways. Our previous studies demonstrated that disruption of lipid rafts by cholesterol-depletion reagent blocked the agonistinduced internalization of OPRM1 and G protein-dependent signaling. The present study demonstrated that reduction of cholesterol level decreased and culturing cells in excess cholesterol increased the agonist-induced internalization and desensitization of OPRM1, respectively. Further analyses indicated that modulation of cellular cholesterol level did not affect agonist-induced receptor phosphorylation but did affect membrane translocation of ␤-arrestins. The translocation of ␤-arrestins was blocked by cholesterol reduction, and the effect could be reversed by incubating with cholesterol. OptiPrep gradient separation of lipid rafts revealed that excess cholesterol retained more receptors in lipid raft domains and facilitated the recruitment of ␤-arrestins to these microdomains upon agonist activation. Moreover, excess cholesterol could evoke receptor internalization and protein kinase C-independent extracellular signal-regulated kinases activation upon morphine treatment. Therefore, these results suggest that cholesterol not only can influence OPRM1 localization in lipid rafts but also can effectively enhance the recruitment of ␤-arrestins and thereby affect the agonist-induced trafficking and agonist-dependent signaling of OPRM1.

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Section: Discussionmentioning

confidence: 56%

Cholesterol Regulates μ-Opioid Receptor-Induced β-Arrestin 2 Translocation to Membrane Lipid Rafts

Qiu¹,

Wang²,

Law³

et al. 2011

Mol Pharmacol

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“…The enzyme-linked immunosorbent assay (ELISA) was performed as described previously [20,22] to determine the level of surface-expressed HA-tagged NK1-R. Briefly, HEK-NK1-R+pEYFP-actin transfected cells from passage numbers 3, 9 and 15 were seeded out at a density of 1×10 5 cells per well in 24-well plate.…”

Section: Elisa Of Surface-expressed Nk1-rmentioning

confidence: 99%

“…The eukaryotic ribosomal (r) 18s RNA (ABI PRISM TaqMan ® Pre-developed Assay Reagent Ribosomal RNA control PN 4310893E) was used as an endogenous control. The real-time polymerase chain reaction (PCR) was then performed with TaqMan universal PCR Master Mix (Applied Biosystems) in a final reaction volume of 20 μl in an AbiPrism 7000 Sequence Detection System as previously described [20]. PCR amplification was carried out for 40 cycles.…”

Section: Quantitative Real-time Reverse Transcription/polymerase Chaimentioning

confidence: 99%

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Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged β-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R)

Hrovat¹,

Zavec

Pogačnik³

et al. 2010

Cellular and Molecular Biology Letters

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Abbreviations used: 7TM -seven transmembrane receptor; B max -max. receptor number; BSA -bovine serum albumin; Ct -threshold cycle; DMEM -Dulbecco's modified Eagles's Medium; DNA -deoxyribonucleic acid; D-PBS -Dulbecco's phosphate-buffered saline; ELISA -enzyme-linked immunosorbent assay; F-actin -filamentous actin; FCAflow cytometry analysis; FCS -flow cytometry sorting; GFP -green fluorescent protein; GPCR -G-protein coupled receptor; HA -hemagglutinin; HANK1-R -N-terminally HAtagged human neurokinin-1 receptor; HEK-293 -human embryonic kidney cells; HIFCSheat inactivated fetal calf serum; HRP -horseradish peroxidase; IC 50 -50% inhibitory concentration; IP1 -inositol phosphate 1; mRNA -messenger ribonucleic acid; NK1-Rneurokinin type 1 receptor; pEYFP -enhanced yellow fluorescent protein; PN -passage number; RT -reverse transcription; RT-PCR -quantitative real-time reverse transcription / polymerase chain reaction; SP -substance P; TMB -tetramethylbenzidine; WT -wildtype Abstract: This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative realtime reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total β-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor-and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFPactin:endogenous β-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidinestained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.

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“…These are lipid rich regions composed of heterogeneously and laterally segregated classes of freely flowing lipid and cholesterol variants assembled via van der Waals interactions, that provide an organizational platform for GPCRmediated signal transduction from the cell surface (Xu et al, 2006;Hanzal-Bayer & Hancock, 2007;Kubale et al, 2007;Morris et al, 2008;Patel et al, 2008). Several recent reports have demonstrated differential localization of PARs in these plasma membrane structures.…”

Section: Differential Plasma Membrane Localization Of Parsmentioning

confidence: 99%

Novel agonists & antagonists for protease-activated receptor 2 and C3a receptor

Yau¹

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About 30-40% of all drugs currently in the market place target G-protein coupled receptors, however of the 900 plus GPCRs predicted to exist only about 30 are targeted by approved drugs. Two novel human GPCRs were investigated in this thesis, namely protease-activated receptor 2 (PAR2) and C3a receptor (C3aR). These membranebound receptors are implicated in a variety of diseases in which inflammation is a common component, making them attractive targets for drugs that can treat such disorders. This thesis describes the rational design, synthesis and in vitro assay of ligands targeting these two GPCRs and they will become useful probes for investigating the pathology of inflammatory disorders and may lead to possible future treatments.Chapter 1 of this thesis summarises peptidic and non-peptidic ligands for proteaseactivated receptor and C3a receptor reported in the literature to date, together with their known physiological effects.Chapter 2 examines structure-activity relationships for analogues of the only known potent PAR2 antagonist (GB88) discovered at IMB. The chemical features of GB88 were investigated separately by dividing the molecule into fragments, which were independently varied to study the effect of structure on agonist/antagonist function of the ligands. A potent and selective PAR2 agonist (132) was developed with EC 50 of 0.4 µM in calcium release assays on HT29 cells. Agonist 132 has comparable agonist potency and better ligand efficiency to 2f-LIGRLO-NH 2 , which was previously the most potent PAR2 agonist prior to these studies. In addition, a new PAR2 antagonist was developed (167), which was 2-fold more potent than GB88 in the calcium mobilisation assay.Chapter 3 describes the non-peptidic PAR2 agonist (GB110) and SAR studies for the truncation of this compound that led to the development of new PAR2 antagonists (192, 209, 211, 212). These ligands inhibited the activation of PAR2 induced by 1 µM 2f-LIGRLO-NH 2 with IC 50 0.4-0.6 µM in the calcium release assay.Chapter 4 evaluates the anti-inflammatory properties of newly developed PAR2antagonists (133, 159, 167, 192 -from Chapters 2 & 3) in both in vivo and in vitro experiments. The most potent PAR2 antagonists (167 and 192) were stable in rat plasma and rat liver homogenates and were able to inhibit PAR2 activation induced by peptide agonist 2f-LIGRLO-NH 2 as well as the endogenous activator, trypsin. These iv antagonists have been demonstrated to be anti-inflammatory agents, inhibiting proinflammatory cytokine release (IL6 and TNF-α) and were effective in relieving joint inflammation in rat models of arthritis.Chapter 5 explores various aromatic heterocycles that confer a turn conformation to ligands and this mimics the turn conformation observed for the C-terminus of C3a in its crystal structure. The study investigates how the hydrogen bonding capabilities of heteroatoms in each heterocycle affect the binding and functions of the ligands.Increasing the hydrogen bond acceptor properties of the heterocycle improves binding affinity...

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Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with β-arrestin 2

Cited by 26 publications

References 59 publications

Cholesterol Regulates μ-Opioid Receptor-Induced β-Arrestin 2 Translocation to Membrane Lipid Rafts

Cholesterol Regulates μ-Opioid Receptor-Induced β-Arrestin 2 Translocation to Membrane Lipid Rafts

Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged β-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R)

Novel agonists & antagonists for protease-activated receptor 2 and C3a receptor

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