Thrombopoietin receptor‐based protein–protein interaction screening (THROPPIS)

Horikawa, Makiko; Kakiuchi, Yosuke; Kashima, Daiki; Ogawa, Kenichiro; Kawahara, Masahiro

doi:10.1002/bit.27975

Cited by 2 publications

(3 citation statements)

References 56 publications

(48 reference statements)

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“…Plat-E cells were seeded in 6-well plates a day before transfection. The cells were transfected with the retroviral plasmids using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, MA) as described previously 15 . Two days later, each well of a 24-well plate was coated with 25 μg/ml Retronectin (Takara Bio, Shiga, Japan), blocked with 2% BSA/PBS, and washed with PBS at room temperature.…”

Section: Retroviral Transductionmentioning

confidence: 99%

“…PPI between the target proteins triggers receptor dimerization and subsequent growth signaling, leading to cell growth as a readout. Since we employed c-Kit or thrombopoietin receptor as the intracellular signaling domain, we named c-Kit-based PPI screening (KIPPIS) [12][13][14] or thrombopoietin receptor-based PPI screening (THROPPIS) 15 , respectively. KIPPIS and THROP-PIS constitutively express chimeric receptors through stable retroviral transduction, which prevents off-target signals compared to the conventional PPI detection systems using transient overexpression.…”

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confidence: 99%

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A growth-based platform for detecting domain–peptide interactions in the cytoplasm of mammalian cells

Kimura

Kashima

Kawahara

2022

Sci Rep

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Development of a method for detecting protein–protein interactions (PPIs) in living cells is important for therapeutic drug screening against various diseases including infectious diseases. We have recently developed a method named SOS localization-based interaction screening (SOLIS), in which we designed membrane-anchored and SOS-fused chimeric proteins, whose PPI-dependent association triggers membrane localization of the SOS-fused chimeric protein, activates the Ras/MAPK pathway, and induces cell growth. While SOLIS was able to detect relatively strong PPIs, further sensitivity was required for detecting intracellular endogenous PPIs typically having a micromolar order of dissociation constant (Kd). Here we develop high-sensitive SOLIS (H-SOLIS) that could universally detect PPIs with lower affinities. In order to improve the sensitivity, H-SOLIS introduces a heterodimeric helper interaction, in which addition of a small-molecule helper ligand could accommodate association of the two chimeric proteins and regulate the sensitivity. Four types of domain–peptide interactions having known Kd values are employed to examine the versatility and detection limit of H-SOLIS. Consequently, the heterodimer-inducible helper ligand dramatically enhances detection sensitivity, lowering the detection limit to a ten-micromolar order of Kd. Thus, H-SOLIS could be a platform to detect disease-related domain–peptide interactions for drug discovery screening.

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Section: Retroviral Transductionmentioning

confidence: 99%

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confidence: 99%

A growth-based platform for detecting domain–peptide interactions in the cytoplasm of mammalian cells

Kimura

Kashima

Kawahara

2022

Sci Rep

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“…The transmembrane type has more choices of ligands, and the membrane raft facilitates association between the receptor chains. On the contrary, the intracellular type has a higher degree of conformational freedom in the dimer/oligomer structure after ligand binding 22 . However, little is known about which receptor type signals more efficiently for inducing a certain cell fate.…”

Section: Introductionmentioning

confidence: 99%

Synthetic receptor scaffolds significantly affect the efficiency of cell fate signals

Umene,

Kawahara

2024

Sci Rep

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Mimicry of receptor functions by designing synthetic receptors would be one of the recently hot research trends in cell engineering. While several types of synthetic receptors have been designed to induce desired cell fates in response to external stimuli, little is known about which receptor type signals more efficiently for inducing a certain cell fate. In this study, we compared the performance of three types of synthetic receptor scaffolds, i.e. myristoylated, cytosolic, and transmembrane types that signal through JAK-dependent phosphorylation of tyrosine motifs to transduce growth signaling. As a result, the phosphorylation levels of JAK and subsequent downstream signaling molecules were significantly maintained in the cytosolic type receptors, leading to more efficient cell growth than the other types. In contrast, the phosphorylation levels of JAK decreased in a motif-dependent manner in the transmembrane type receptors. Although various studies on receptor engineering based on domain or motif engineering have been reported, to our knowledge this study is the first to demonstrate that synthetic receptor scaffolds significantly affect the efficiency of cell fate signals. These findings are important for both receptor biology and receptor engineering, providing guidelines for rationally designing synthetic receptors that can transduce as efficient signaling as possible.

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Thrombopoietin receptor‐based protein–protein interaction screening (THROPPIS)

Cited by 2 publications

References 56 publications

A growth-based platform for detecting domain–peptide interactions in the cytoplasm of mammalian cells

A growth-based platform for detecting domain–peptide interactions in the cytoplasm of mammalian cells

Synthetic receptor scaffolds significantly affect the efficiency of cell fate signals

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