Cell‐Surface Expression Levels Are Important for Fine‐Tuning the Performance of Receptor Tyrosine Kinase‐Based Signalobodies

Abstract

As receptor tyrosine kinases (RTKs) play important roles in cell-fate control of various cell types, engineered RTKs that could respond to inexpensive ligands might drastically reduce the cost of producing desired cells for various applications in regenerative medicine. We developed several engineered RTKs named "signalobodies" in which the ligand-recognition domain of RTKs is replaced by single-chain Fv for enabling recognition of a specific antigen. However, the remaining concern was the dysregulation of antigen-dependent on/off signaling of the signalobodies. This study aims at fine-tuning the performance of the signalobodies based on three RTKs (fibroblast growth factor receptor 1, insulin receptor, and c-fms). To this end, the cell-surface expression levels of the RTK-based signalobodies were altered by locating their genes either upstream or downstream of the internal ribosomal entry site, and by inserting 1 to 3 alanine residue(s) at the intracellular juxtamembrane region. As a result, while the signaling response was different among the three signalobodies, the antigen-dependent on/off regulation became tighter when the cell-surface expression levels of the signalobodies were lowered. Therefore, we successfully developed a method to diminish the leaky signaling of RTK-based signalobodies, which will be important for establishing the signalobody-based platform technology that can produce cells of interest for regenerative medicine.

Cell fate‐inducing CARs orthogonally control multiple signaling pathways

“…The amino acid sequences of cfiCARs are provided in Supplementary Information (Supplementary Figure S1–S6). The dinitrophenol (DNP)‐responsive gp130‐CAR and signal transducers and activators of transcription (STAT)3‐CAR genes were encoded in a retroviral plasmid pMK‐stuffer‐ internal ribosomal entry site (IRES)‐Puro R , [ 19 ] whereas another DNP‐responsive Fas‐CAR gene was encoded in a doxycycline‐inducible retroviral plasmid pRetroX‐Tight‐Puro R (Takara Bio, Shiga, Japan). The fluorescein (FL)‐responsive receptor activator of nuclear factor κB (RANK)‐CAR and Shc‐CAR genes were encoded in a retroviral plasmid pMK‐stuffer‐IRES‐Neo R , [ 28 ] whereas another FL‐responsive Fms‐CAR gene was encoded in a retroviral plasmid pMK‐stuffer‐IRES‐Blast R .…”

“…Murine interleukin (IL‐3)‐dependent Ba/F3 and retroviral packaging Plat‐E cell lines were cultured as described previously. [ 19 ] A Ba/3K cell line, [ 23 ] which is a Ba/F3 variant constitutively expressing the Tet‐On 3G transactivator of Tet‐On 3G Inducible Expression System (Takara Bio), was cultured in RPMI 1640 medium with 1 ng mL −1 murine IL‐3 (Kaico, Fukuoka, Japan), 800 μg mL −1 G418 (Calbiochem, La Jolla, CA), and 10% Tet system‐approved fetal bovine serum (Takara Bio). Various concentrations of doxycycline (Takara Bio) were further added to Ba/3K transductants for inducible expression of chimeric receptors.…”

“…Western blotting was performed as described previously. [ 19 ] We used the following rabbit primary antibodies: anti‐phospho‐c‐Jun N‐terminal kinase (JNK) (T183/Y185) (Cell Signaling Technology, Danvers, MA), anti‐JNK (Santa Cruz Biotechnology, Santa Cruz, CA), anti‐phospho‐STAT3 (Y705) (Cell Signaling Technology), anti‐STAT3 (Santa Cruz Biotechnology), anti‐phospho‐Shc (Y239/240) (Cell Signaling Technology), anti‐Shc (Santa Cruz Biotechnology), anti‐phospho‐MEK1/2 (S217/221) (Cell Signaling Technology), anti‐MEK1/2 (Cell Signaling Technology), anti‐cleaved caspase 3 (Cell Signaling Technology), anti‐Flag tag (Thermo Fisher Scientific), anti‐HA tag (Bethyl Laboratories, Montgomery, TX), anti‐Myc tag (Bethyl Laboratories), anti‐β‐actin (Santa Cruz Biotechnology), and anti‐GAPDH (Santa Cruz Biotechnology).…”

“…To this end, we constructed a series of chimeric receptors in which the extracellular domains of these cytokine receptors were replaced with scFv. As a result, we succeeded in developing CARs that control the four fundamental cell fates, namely proliferation-, differentiation-, migration-, and apoptosis-inducing CARs (piCAR, [18,19] diCAR, [12,20] miCAR, [21,22] and aiCAR, [23,24] respectively).…”

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

“…To investigate whether other receptors in the same family members could be used for our PPI detection system, we aimed to test EpoR and gp130, which are Type I cytokine receptors, and EGFR, c-fms, and IR, which are receptor tyrosine kinases (Figure 4a). These receptors have already proved to induce cell growth when activated in the plasma membrane of Ba/F3 cells in the previous research works (Nakabayashi et al, 2017(Nakabayashi et al, , 2013Tanaka et al, 2009). We substituted the intracellular domain of these receptors for that of TPOR in the chimeric receptors incorporating PB1n and PA as representative POIs, and investigated whether the chimeric receptors could detect PPIs in the cytoplasm (Figure 4b).…”