Refining minimal engineered receptors for specific activation of on-target signaling molecules

Differential localization of MERs revealed significantly different signaling intensities

The MERs constructed in our previous study consisted of a myristoylation signal sequence, a dimerization domain FKBP_F36V, the JAK-binding domain of c-mpl, and tyrosine motifs, hereafter designated as a Myr type¹⁴. The Myr-type MERs showed off-target activation of STAT5 even in the absence of AP20187, which is a synthetic dimerizer ligand for FKBP_F36V. This off-target activation of STAT5 may be due to either direct activation of STAT5 by JAK^15,16 or nonspecific binding of STAT5 to the tyrosine motifs within the MERs. In this study, we constructed a series of MERs to examine these possibilities (Fig. 1a). First, we incorporated a STAT3-binding motif or its tyrosine-to-phenylalanine mutant as a tyrosine motif of MERs, aiming to compare the signaling properties in parallel. In addition, since off-target activation of STAT5 in the absence of ligand stimulation may be caused by increased frequency of collisions of MERs through membrane localization, we also constructed a non-membrane-localized cytoplasmic (Cyt) type and in contrast a transmembrane (TM) type. As for the extracellular domain of the TM type, the EpoR D2 domain in addition to FKBP_F36V was incorporated because our previous studies showed that receptor constructs using the EpoR D2 domain resulted in sufficient expression and signaling of various antibody-receptor chimeras^{17,18,19,20,21,22,23,24,25,26,27}.

Differential localization of MERs revealed significantly different signaling intensities. (a) The molecular architecture of the constructed MERs. The myristoylated (Myr), cytoplasmic (Cyt), and transmembrane (TM) type receptors were designed. The STAT3-binding motif (STAT3) or its tyrosine-to-phenylalanine mutant (STAT3F) were incorporated into the signaling domain of the MERs. (b) Signaling analysis. Parental Ba/F3 cells and the transductants were stimulated with no ligand (-), 50 nM AP20187 (+), or 1 ng/mL IL-3 (IL3). Blots with the phospho-specific antibodies detected activation levels of signaling molecules, whereas blots with anti-Myc tag detected expression levels of the MERs (n = 1). Blots with anti-whole signaling molecules and anti-GAPDH serve as loading controls (n = 1). Uncropped blot images are provided in Supplementary Information (Supplementary Fig. 6).

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IL-3-dependent Ba/F3 cells were transduced with retroviral vectors encoding these MERs, and stable transductants were obtained by antibiotic selection. The cells were depleted in IL-3-free medium and stimulated with AP20187 to activate the MERs (Fig. 1b). Western blotting confirmed expression of all three types of the MERs at the expected molecular sizes. Signaling analysis showed that the Myr and Cyt types activated the target signaling molecule STAT3, with the Cyt type being particularly efficient, whereas the TM type was unable to activate STAT3. The off-target activation of STAT5 was ligand-independent in the Myr and TM types. The Cyt type had the strongest off-target activation of STAT5 with ligand stimulation, but the weakest off-target activation of STAT5 without ligand stimulation. In addition, even when the STAT3-binding motif was functionally knocked out by the tyrosine-to-phenylalanine mutation, off-target activation of STAT5 still occurred, indicating that off-target activation of STAT5 is not due to the tyrosine motif. These results suggest that off-target activation of STAT5 occurs due to enhanced receptor–receptor association through membrane localization in the Myr and TM types, whereas off-target activation of STAT5 occurs in the Cyt type when JAK is activated in a ligand-dependent manner.

Two tyrosines in the JAK-binding domain do not contribute to off-target activation of STAT5

Although the experiments in the previous section revealed that off-target activation of STAT5 was not due to the tyrosine motif, another possibility is that the two tyrosine residues in the JAK-binding domain may function as cryptic tyrosine motifs and recruit STAT5 upon phosphorylation. To test this possibility, one or both of the two tyrosine residues were mutated to phenylalanine in the Myr- and Cyt-type MERs (Fig. 2a). Signaling analysis showed that even when both tyrosine residues were mutated to phenylalanine, on-target activation of STAT3 and off-target activation of STAT5 occurred at almost the same levels as those observed in the intact constructs (Fig. 2b). Therefore, the results proved that the two tyrosine residues neither affect the activation levels of JAK nor involve off-target activation of STAT5. However, since the two tyrosine residues might cause off-target activation of signaling molecules other than STAT5, hereafter we utilized the mutated JAK-binding domain in which both tyrosine residues were mutated to phenylalanine. With regard to the receptor type, we chose the Cyt type, which attained high phosphorylation levels of the on-target signaling molecule.

Two tyrosines in the JAK-binding domain do not contribute to off-target activation of STAT5. (a) The molecular architecture of the constructed MERs. One or both of the two tyrosine residues in the JAK-binding domain were mutated to phenylalanine in the Myr- and Cyt-type MERs. The STAT3-binding motif (STAT3) was incorporated into the signaling domain of the MERs. (b) Signaling analysis. Parental Ba/F3 cells and the transductants were stimulated with no ligand (-), 50 nM AP20187 (+), or 1 ng/mL IL-3 (IL3). Blots with the phospho-specific antibodies detected activation levels of signaling molecules, whereas blots with anti-Myc tag detected expression levels of the MERs (n = 1). Blots with anti-whole signaling molecules and anti-GAPDH serve as loading controls (n = 1). Uncropped blot images are provided in Supplementary Information (Supplementary Fig. 7).

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Box1 point mutations modulate on-target/off-target ratio

In the experiments described in the previous section, off-target activation of STAT5 was observed even when all tyrosine residues in the MERs were removed, indicating that it was due to direct activation of STAT5 by JAK. Therefore, it is necessary to lower the off-target activation level of STAT5 while maintaining on-target activation levels by modulating activation modes of JAK. The amino acid residues in Box1 of the JAK-binding domain are known to be critical for JAK binding^16,28,29,30. Therefore, we aimed to screen for Box1 point mutants with high on-target/off-target ratios. Six highly conserved consensus amino acid residues in Box1²⁸ were point-mutated to alanine (Fig. 3a), and transductants expressing each of the mutants were subjected to signaling analysis. As a result, when W, 1P, 2P, and 3L were mutated to alanine, on-target activation of STAT3 was significantly reduced, while off-target activation of STAT5 was eliminated (Fig. 3b). On the other hand, when 1L was mutated to alanine, on-target activation of STAT3 was almost maintained, and off-target activation of STAT5 was almost abolished. When 2L was mutated to alanine, both on-target activation of STAT3 and off-target activation of STAT5 were reduced. To confirm these observations quantitatively, we performed experiments to obtain additional 4 gel blots for pSTAT3/STAT3 and pSTAT5/STAT5 and quantified the bands (Fig. 3c). As a result, the on-target/off-target ratios changed depending on the Box1 mutants, among which the 1L→A mutant gave the highest value. Furthermore, the difference between the intact and 1L→A mutant was statistically significant. Thus, the 1L→A mutant revealed desirable signaling properties with a better on target/off target ratio than the intact.

Box1 point mutations modulate on-target/off-target ratio. (a) The molecular architecture of the constructed MERs. Six highly conserved consensus amino acid residues in Box1 (filled in yellow) were point-mutated to alanine in the Cyt-type MER with two tyrosine-to-phenylalanine mutations of the JAK-binding domain. The STAT3-binding motif (STAT3) was incorporated into the signaling domain of the MERs. (b) Signaling analysis. Parental Ba/F3 cells and the transductants were stimulated with no ligand (-), 50 nM AP20187 (+), or 1 ng/mL IL-3 (IL3). Blots with the phospho-specific antibodies detected activation levels of signaling molecules, whereas blots with anti-Myc tag detected expression levels of the MERs (n = 1). Blots with anti-whole signaling molecules and anti-GAPDH serve as loading controls (n = 1). Uncropped blot images are provided in Supplementary Information (Supplementary Fig. 8). (c) Quantification of the on-target/off-target ratios from the blotting data. The uncropped blot images of additional 4 gel blots for pSTAT3/STAT3 and pSTAT5/STAT5 are provided in Supplementary Information (Supplementary Fig. 9). The phosphorylation level of each signaling molecule with AP20187 stimulation was normalized by the value of IL-3-stimulated parental cells, and further normalized by the expression level in order to enable comparison among the multiple gel blots. The on-target/off-target ratios were calculated by dividing the normalized pSTAT3 values by the normalized pSTAT5 values. The data are represented as mean ± SD (n = 5). One-way ANOVA followed by post-hoc Bonferroni’s multiple-comparison test was used for comparing the means of the groups. ***p < 0.001; **p < 0.01; n.s., not significant versus intact.

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The refined MER enables analysis of signaling properties of arbitrary tyrosine motifs

We aimed to incorporate a series of tyrosine motifs (with putative specificity toward STAT1 to 6 and Shc) into the refined Cyt-type MER variant (JAK-bd 1F, 2F / Box1 1L→A) and evaluate the signaling properties of each tyrosine motif. The STAT1-, STAT3-, STAT5-, and Shc-binding motifs, which can preferentially activate the corresponding signaling molecules, are the same as those used in our previous study^10,14. On the other hand, the STAT2-, STAT4-, and STAT6-binding motifs are chosen based on literature and used for the first time in this study. The STAT2-binding motif is derived from IFNAR2 and has been shown to activate not only STAT2 but also STAT1 and STAT3³¹. The STAT4- and STAT6-binding motifs are derived from IL-12Rβ2 and IL-4Rα, respectively, but specificities of the motifs are unknown^32,33.

We established stable transductants expressing the MERs incorporating these motifs and examined their signaling properties by Western blotting. Consequently, corresponding bands were observed correctly for the phospho-STAT1, 3, 5, 6, and Shc, whereas no corresponding bands were observed at the expected molecular sizes for the phospho-STAT2 and 4 (Fig. 4). The specificities of the putative STAT2- and STAT4-binding motifs were insufficient, activating all of STAT1, 3, 5, and 6. In contrast, the putative STAT1-, 3-, 5-, 6-, and Shc-binding motifs activated the corresponding signaling molecules more specifically, although non-specific activation of the other signaling molecules was observed at weak levels. Only the Shc-binding motif activated Akt and MEK, indicating that the STATs-binding motifs did not recruit adapter molecules that activate the Akt pathway and/or the Ras/MAPK pathway. Thus, the STATs-binding motifs revealed to be specific to STATs, but could be of a promiscuous nature within STATs depending on the motifs.

The refined MER enables analysis of signaling properties of arbitrary tyrosine motifs. Parental Ba/F3 cells and the transductants with putative STATs- and Shc-binding motifs in the refined MER were stimulated with no ligand (-), 50 nM AP20187 ( +), or 1 ng/mL IL-3 (IL3). Blots with the phospho-specific antibodies detected activation levels of signaling molecules, whereas blots with anti-Myc tag detected expression levels of the MERs (n = 1). Blots with anti-whole signaling molecules and anti-GAPDH serve as loading controls (n = 1). Uncropped blot images and a graph qualitatively illustrating the results are provided in Supplementary Information (Supplementary Figs. 10 and 11).

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Phenotypic assays revealed differential cell fate-inducing properties among the motifs

To analyze cell fate-inducing properties of the MERs incorporating the tyrosine motifs, we performed phenotypic assays on proliferation, migration, and motility. A cell proliferation assay showed that ligand-dependent cell proliferation was strongly induced by the STAT3-, STAT5-, and Shc-binding motifs, weakly induced by the STAT4- and STAT6-binding motifs, and barely induced by the STAT1- and STAT2-binding motifs (Fig. 5a). A cell migration assay using transwell plates showed that cell migration was induced in response to a positive gradient of the ligand only by the Shc-binding motif (Fig. 5b). To verify whether the cell migration induced by the Shc-binding motif was chemotactic or chemokinetic, four gradient patterns were created in the top and bottom chambers with and without the ligand. Consequently, cell migration occurred even when the ligand was added to the top chamber, demonstrating a chemokinetic nature of the cell migration (Fig. 5c). Time-lapse observation of cell motility in a horizontal chamber showed that cell motility was significantly enhanced in response to the ligand only by the Shc-binding motif (Supplementary Movies 1 to 16). In the signaling analysis shown in Fig. 4, the Akt and Ras/MAPK pathways were activated only by the Shc-binding motif, which coincides the results of cell migration and motility assays. Taken together, activation of the Ras/MAPK and Akt pathways was important for cell migration and motility, while the JAK/STAT pathway was not involved in these cellular phenotypes. These results demonstrate that the refined MER is useful for analyzing the signaling properties of tyrosine motifs and precisely inducing desired cellular phenotypes.

Phenotypic assays revealed differential cell fate-inducing properties among the motifs. (a) Cell proliferation assay. Cells were cultured with the indicated concentrations of AP20187 for 3 days, and cell proliferation levels were measured by a colorimetric assay. The data are represented as mean ± SD (n = 3, triplicate cultures). (b, c) Cell migration assay. Cells (3 × 10⁵) were added to the top chambers of 24-well transwell plates. Cell migration to the bottom chambers after incubation for 5 h was measured by counting with a flow cytometer. The data represent mean ± SD (n = 3, triplicate wells). (b) The ligand AP20187 (50 nM) was added to the bottom chambers to create positive gradients. (c) The cells expressing the MER with the Shc-binding motif were subjected to this assay. AP20187 (50 nM) was added to the bottom and/or top chambers to create positive (top/bottom: − / +), negative (+ / −), and no (− / − and + / +) gradients.

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• Source: https://www.nature.com/articles/s41598-024-81259-4