Tools and Techniques to Study Ligand–Receptor Interactions and Receptor Activation by TNF Superfamily Members

Schneider, Pascal; Willen, Laure; Smulski, Cristian R.

doi:10.1016/b978-0-12-801430-1.00005-6

Cited by 41 publications

(60 citation statements)

References 19 publications

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“…Receptor-Ligand Interaction ELISA-The direct binding of FLAG-tagged ligands to immobilized receptor-Fcs, either adsorbed to or captured via anti-human antibodies in 96-well immunoplates, was revealed with biotinylated anti-FLAG M2 antibody (Sigma) and horseradish-coupled streptavidin as described previously in detail (22). For the competition ELISA, titrated amounts of untagged ligands were added to immobilized receptor-Fcs for 30 min and followed, without intermediate washing step, by an empirically determined fixed and nonsaturating concentration of FLAG-tagged ligand sufficient to generate ϳ80% of the maximal signal in the absence of competitor (22).…”

Section: Methodsmentioning

confidence: 99%

“…For the competition ELISA, titrated amounts of untagged ligands were added to immobilized receptor-Fcs for 30 min and followed, without intermediate washing step, by an empirically determined fixed and nonsaturating concentration of FLAG-tagged ligand sufficient to generate ϳ80% of the maximal signal in the absence of competitor (22).…”

Section: Methodsmentioning

confidence: 99%

“…Belimumab (registered trade name Benlysta) was purchased from the Pharmacy of Lausanne University Hospital. Other recombinant proteins were produced essentially as described (21,22).…”

Section: Methodsmentioning

confidence: 99%

“…FACS Analyses-293T cells co-transfected with EGFP and receptor-GPI expression plasmids were stained with titrated amounts of FLAG-or Fc-tagged ligands, whose binding was revealed with appropriate phycoerythrin-coupled secondary reagents, as described (22,30). Cells were analyzed using FACScan or Accuri 6 flow cytometers (BD Biosciences) and FlowJo software (TreeStar, Ashland, OR).…”

Section: Methodsmentioning

confidence: 99%

“…Similarly, Jurkat JOM2 BAFFR:Fas cell lines (cl21) were generated as described in detail elsewhere by retrovirus-mediated transduction of a BAFFR:Fas chimeric receptor, puromycin selection, and screening of clones for their selective ability to respond to Fc-BAFF by apoptotic cell death (22). Reporter cells were incubated for 16 h with the indicated concentrations of ligands, after which time cell viability was measured by the phenazine methosulfate/3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay essentially as described (22). FLAGtagged ligands were used in the presence of 1 g/ml anti-FLAG M2 antibody.…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties

Schuepbach‐Mallepell

Das

Willen

et al. 2015

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Belimumab (registered trade name Benlysta) was purchased from the Pharmacy of Lausanne University Hospital. Other recombinant proteins were produced essentially as described (21,22).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties

Schuepbach‐Mallepell

Das

Willen

et al. 2015

Journal of Biological Chemistry

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Bio‐nanocapsule‐based scaffold improves the sensitivity and ligand‐binding capacity of mammalian receptors on the sensor chip

Iijima

Yoshimoto

Niimi

et al. 2016

Biotechnology Journal

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Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand-binding assays using immobilized receptors. A bio-nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A-derived immunoglobulin G (IgG) Fc-binding Z domains (denoted as ZZ-BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc-fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ-BNC on the sensor chip of quartz crystal microbalance (ZZ-BNC-coating). When compared with direct adsorption and protein A-coating, the sensor chip showed higher sensitivity (∽46- and ∽165-fold, respectively) and larger ligand-binding capacity (∽4- and ∽18-fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ-BNC-coating, strongly suggesting that ZZ-BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand-binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ-BNC and Fc-fused receptors could significantly improve the function of ligand-binding assays.

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Porphyrin Derivatives Inhibit the Interaction between Receptor Activator of NF‐κB and Its Ligand

et al. 2017

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Receptor activator of NF-κB (RANK), a member of the TNF-receptor superfamily, plays an important role in bone resorption and stimulates immune and epithelial cell activation. Denosumab, a human monoclonal antibody that blocks the RANK ligand (RANKL), is approved for the treatment of osteoporosis and bone metastasis. However, a small molecule that inhibits the RANK-RANKL interaction would be beneficial to decrease cost and to facilitate treatments with orally available therapeutic agents. Herein we report the discovery of the first nonpeptidic inhibitors of RANK-RANKL interactions. In screening a chemical library by competitive ELISA, the porphyrin verteporfin was identified as a hit. Derivatives were screened, and the chlorin-macrocycle-containing pheophorbide A and purpurin 18 were found to bind recombinant RANKL, to inhibit RANK-RANKL interactions in the ELISA, and to suppress the RANKL-dependent activation of model cells and the differentiation of RANK-expressing precursors into osteoclasts. This discovery of a family of small molecules that inhibit RANK activation presents an initial basis for further development of nonpeptidic therapeutic agents targeting the interaction between RANK and RANKL.

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Tools and Techniques to Study Ligand–Receptor Interactions and Receptor Activation by TNF Superfamily Members

Cited by 41 publications

References 19 publications

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties

Bio‐nanocapsule‐based scaffold improves the sensitivity and ligand‐binding capacity of mammalian receptors on the sensor chip

Porphyrin Derivatives Inhibit the Interaction between Receptor Activator of NF‐κB and Its Ligand

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