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Designing motif-engineered receptors to elucidate signaling molecules important for proliferation of hematopoietic stem cells Shuta Ishizuka, Chen-Yi Lai, Makoto Otsu, Hiromitsu Nakauchi, Teruyuki Nagamune, and Masahiro Kawahara ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00163 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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ACS Synthetic Biology

Designing motif-engineered receptors to elucidate signaling molecules important for proliferation of hematopoietic stem cells

Shuta Ishizuka1, Chen-Yi Lai2, Makoto Otsu2, Hiromitsu Nakauchi3, Teruyuki Nagamune1, Masahiro Kawahara1,*

1

Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of

Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; 2

Division of Stem Cell Processing / Stem Cell Bank, Center for Stem Cell Biology and

Regenerative Medicine, Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; 3

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of

Medicine, Stanford, CA 94305, USA

*

Corresponding author:

Telephone: +81-3-5841-7290; fax: +81-3-5841-7335; e-mail: [email protected]

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Abstract The understanding of signaling events is critical for attaining long-term expansion of hematopoietic stem cells ex vivo. In this study, we aim to analyze the contribution of multiple signaling molecules in proliferation of hematopoietic stem cells. To this end, we design a bottom-up engineered receptor with multiple tyrosine motifs, which can recruit multiple signaling molecules of interest. This is followed by a top-down approach, where one of the multiple tyrosine motifs in the bottom-up engineered receptor is functionally knocked out by tyrosine-to-phenylalanine mutation. The combination of these two approaches demonstrates the importance of Shc in cooperation with STAT3 or STAT5 in the proliferation of hematopoietic stem cells. The platform developed herein may be applied for analyzing other cells and/or other cell fate regulation systems.

Keywords: chimeric receptor, motif engineering, signal transduction, hematopoietic stem cell, artificial ligand


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Cells respond to extracellular signals via transmembrane receptors, which initiate intracellular signal transduction to control cell fate.1-4 Practically, proliferation of stem cells and their differentiation into a specific lineage of interest need to be regulated to attain efficient production of desired cells for applications in regenerative medicine. While embryonic stem cells and several tissue-specific stem cells can be maintained ex vivo,5-7 the long-term ex vivo expansion of hematopoietic stem cells (HSCs) is still challenging.8,9 The proliferation of HSCs is promoted by multiple cytokine combinations,10,11 such as stem cell factor (SCF) and thrombopoietin (TPO), which induce homodimerization and subsequent activation of their specific receptors c-Kit and cMpl, respectively.12,13 These receptors then recruit signaling molecules with Src homology 2 (SH2) domain such as STAT3, STAT5, and Shc via their phosphorylated tyrosine motifs, which activate signaling pathways that induce cell proliferation. Many investigators have analyzed signaling molecules important for proliferation of HSCs. The approaches using constitutively active mutants, knockdown, and inhibitors of specific signaling molecules have suggested the contribution of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT), Ras/mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, and Wnt/β-catenin pathways.1419

However, the contribution of the signaling molecules involved still remains to be elusive. Previously we employed a top-down engineering approach of the wild-type receptor c-Mpl

to identify signaling molecules important for HSC proliferation.20 However, because each motif in c-Mpl recruits multiple signaling molecules, the truncation and mutation of the motifs would affect activation levels of multiple signaling molecules, which prevented from identifying a key signaling molecule for HSC proliferation. Here we propose a novel platform for analyzing the contribution of signaling molecules on cell fate using motif-engineered artificial receptors. This platform is based on the fact that the intracellular domain of type I cytokine receptors is composed of the following two regions; i) a region which associates with a tyrosine kinase JAK and ii) a region containing multiple tyrosine motif sequences that can recruit signaling molecules (Figure 1, a and b).21,22 The deletion of the 3 ACS Paragon Plus Environment


latter region would result in a no-motif receptor. The addition of a tyrosine motif which recruits a specific signaling molecule to the no-motif receptor could activate the signaling molecule of interest (Figure 1b). The engineered receptor intracellular domain is linked to an HA-tagged antifluorescein single-chain Fv via a transmembrane domain so that the resultant chimeric receptor can be activated by homo-oligomerization via an artificial ligand fluorescein-conjugated BSA (BSAFL), which has multiple fluorescein moieties on a BSA molecule. In our previous prototypic study, this bottom-up engineering approach was used for creating 5 motif-grafted chimeras with either of 5 different motifs (the binding motifs for STAT1, STAT3, STAT5, PI3K, and Shc that are all activated by c-Mpl).23 The signaling analyses confirmed that these chimeras can preferentially activate the signaling molecules of interest. When genetically introduced into HSCs, only the chimera with the STAT5-binding motif supported HSC proliferation with the help of SCF-induced c-Kit activation.24 However, the proliferation level was less than that induced by the positive control chimera bearing the wild-type c-Mpl intracellular domain. These results suggest that signaling molecules other than STAT5 may cooperate with STAT5 in the wild-type c-Mpl signaling to efficiently transduce the proliferation signals of HSCs. In this study, we aimed to extend our platform to analyze the contribution of multiple signaling molecules in HSC proliferation. To achieve this, we designed an engineered receptor with multiple tyrosine motifs, which can recruit multiple signaling molecules of interest (Figure 1c). This bottom-up approach can functionally reconstitute the receptor signaling with multiple signaling components. Then, one of the multiple tyrosine motifs was functionally knocked out by tyrosine-tophenylalanine (Y-to-F) mutation for selective ablation of one of the reconstituted signaling components (Figure 1d). This top-down approach reveals the contribution of the selectively ablated signaling molecule when the other signaling molecules are activated. Among signaling molecules of the JAK/STAT signaling pathway, STAT3 is anticipated to play pivotal roles in HSCs.25,26 Besides the JAK/STAT pathway, the Ras/MAPK and PI3K/Akt signaling pathways are also known to be important for proliferation of HSCs.27,28 Shc is an adaptor 4 ACS Paragon Plus Environment

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protein that can activate both Ras/MAPK and PI3K/Akt signaling pathways.29 Taken together, here we chose the STAT3- and Shc-binding motifs as well as the STAT5-binding motif to construct multiple motifs-engineered receptors. In our previous reports, we had created the chimeric receptors whose intracellular domain is either wild-type c-Mpl (“WT”), only the JAK-binding region of c-Mpl (“no motif”), the JAKbinding region plus the STAT5-binding motif (“5”), or the JAK-binding region plus the Shc- and STAT3-binding motifs (“S3”) (Figure 2a).23,30 Here we created in addition the chimeric receptors with three motifs by inserting the STAT5-binding motif at the upstream and downstream of the S3 chimera, resulting in two chimeras (“5S3” and “S35”) (Figure 2a). The multiple motifs of each chimera were linked with a flexible linker (G4S)3, which had been demonstrated to be appropriate for two adjacent motifs to be functional.30 The amino acid sequences for the chimeras are described in Supporting Information (Figure S1 and S2). The gene encoding each chimera was integrated into a retroviral vector which also encodes the puromycin resistance gene and enhanced green fluorescent protein (EGFP) gene so that gene-transduced cells can be not only selected by puromycin but also detected or sorted by flow cytometry. To examine whether these motif-engineered chimeric receptors can activate corresponding signaling molecules, an interleukin (IL)-3-dependent murine pro-B Ba/F3 cell was used as a host cell. Because the growth of this hematopoietic precursor cell line is strictly IL-3-dependent, depletion of IL-3 in the culture medium leads to dephosphorylation of growth-related signaling molecules. When the exogenously expressed receptors are stimulated by their ligand after the depletion, the phosphorylation of signaling molecules is easily detected with high signal-to-noise ratio. Ba/F3 cells were retrovirally transduced with each chimeric receptor (Figure 2a), and stable transductants were obtained after puromycin selection, of which efficiency was confirmed by flow cytometry to detect EGFP expression (Supporting Information, Figure S3). Immunostaining against the appended HA tag followed by flow cytometric analyses revealed that all transductants express the chimeric receptors on the cell surface (Supporting Information, Figure S4). Next, the cells were 5 ACS Paragon Plus Environment


cultured without IL-3 and stimulated with the antigen BSA-FL to activate the chimeric receptors (Figure 2, b and c). Western blot analyses revealed that STAT5 was motif-independently activated by all chimeras including the no-motif chimera, which was consistent in the previous study and may be due to the JAK-dependent STAT5 activation.23 On the other hand, STAT3 and Shc were activated by the chimeras with STAT3- and Shc-binding motifs (the S3, 5S3, and S35 chimeras). To examine the effects of these motif-engineered receptors on the proliferative response of HSCs, murine HSCs (CD34-/lowc-Kit+Sca-1+Lineage marker-) were purified from bone marrow and transduced with retroviral vectors encoding the chimeric receptors (Figure 2d). The gene-transduced cells were sorted according to the EGFP expression (Supporting Information, Figure S5), and seeded in multiwell plates in the presence of SCF alone (activation of endogenous c-Kit), SCF+BSA-FL (activation of endogenous c-Kit and the chimeric receptors), and SCF+TPO (activation of endogenous c-Kit and c-Mpl). After 5 days, the viable cell number was counted on flow cytometry. Surprisingly, the cell proliferation levels induced by S3, 5S3, and S35 exceeded those induced by WT (Figure 2e). This was true with all culture conditions including SCF alone, meaning ligand-independent leaky signaling. In fact, Figure 2b showed that these chimeric receptors (especially S3, 5S3, and S35) appeared to be “switched on” even in the absence of the antigen BSA-FL, supporting the above notion. Despite the signal leak, these chimeric receptors still proved to be able to transduce stronger proliferation signals in HSCs than the others upon stimulation (Figure 2e, SCF + BSA-FL). Taken together, the concurrent activation of Shc, STAT3, and STAT5 is effective for inducing HSC proliferation. Our previous study demonstrated that single motif (STAT5, STAT3, and Shc-binding motif)grafted chimeras (i.e. 5, 3, and S, respectively) cannot induce such high proliferative response of HSCs.24 Therefore, the results in the present study indicate that multiple signaling molecules cooperatively induced efficient HSC proliferation. To identify signaling molecules responsible for the efficient HSC proliferation, we designed a top-down receptor engineering approach, in which one of the three motifs in the 5S3 and S35 chimeras is disrupted by tyrosine-to-phenylalanine (YF) 6 ACS Paragon Plus Environment

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mutation (Figure 3a). The resultant series of chimeras (5YF, SYF, and 3YF mutants derived from the 5S3 and S35 chimeras) lack the capability to recruit STAT5, Shc, and STAT3, respectively. These chimeras were introduced into Ba/F3 cells to yield the stable transductants (Supporting Information, Figure S6). All of the transductants expressed the chimeric receptors on the cell surface (Supporting Information, Figure S7). The signaling analysis showed that the SYF and 3YF mutations successfully ablated the activation of Shc and STAT3, respectively, whereas the 5YF mutation still allowed the STAT5 activation (Figure 3, b, c, d, and e). These chimeras were introduced into HSCs, and their effects on proliferative responses were analyzed after sorting of EGFP-positive cells (Supporting Information, Figure S8). The results demonstrated that the SYF mutants significantly diminished the proliferation levels (Figure 3f). On the other hand, 5YF and 3YF mutants showed little or even enhanced effects on HSC proliferation. These results demonstrate the importance of Shc in cooperation with STAT3 or STAT5 in the proliferative response of HSCs. Because Shc was shown to be important for the HSC proliferation, we examined whether Ras/MAPK and PI3K/Akt signaling pathways, which are downstream of Shc, were activated in an Shc-binding motif-dependent manner. Unexpectedly, MEK, ERK (in the Ras/MAPK pathway), and PI3K (in the PI3K/Akt pathway) were all activated even in the absence of Shc activation (Supporting Information, Figure S9). We suspected that SHP2, which is another important adapter protein that activates both of the Ras/MAPK and PI3K/Akt pathways,31,32 might be involved in the unexpected activation of these pathways. As expected, the SHP2 was also activated even without Shc activation, and the signaling properties coincide well with the activation patterns of MEK, ERK, and PI3K (Figure S9). These results suggest that SHP2 is responsible for the Shc-independent activation of the Ras/MAPK and PI3K/Akt pathways. In spite of the fact that Ras/MAPK and PI3K/Akt pathways in Ba/F3 cells can be activated in the absence of Shc activation, HSC proliferation was prominently restrained when stimulated through receptors lacking the Shc-binding motif, which indicates that signaling downstream of Shc 7 ACS Paragon Plus Environment


is important for HSC proliferation. Intriguingly, previous reports demonstrated that a totally different biological response is generated by the differences of frequency and intensity in the activation of the Ras/MAPK pathway.33-37 Our result here may represent one possibility that Shc and SHP2 may activate the Ras/MAPK and PI3K/Akt pathways with distinct frequencies and/or intensities. In conclusion, we demonstrated that the combination of bottom-up and top-down motifengineering approaches can identify signaling molecules important for HSC proliferation. Using the bottom-up approach, the artificial receptors with multiple motifs induced higher HSC proliferation levels than wild-type c-Mpl. The top-down approach revealed that the activation of Shc+STAT3 or Shc+STAT5 led to efficient HSC proliferation. Novel molecular mechanisms may be unveiled by applying our motif-engineered receptor approach to other cells and/or other cell fate regulation systems.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental procedures; primer sequences;

amino acid sequences of the designed

chimeric receptors; results of gene transduction into Ba/F3 cells and hematopoietic stem cells, immunostaining, and signaling analysis (PDF)

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

Notes The authors declare no competing financial interests. 8 ACS Paragon Plus Environment

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Author Contributions M.K. conceived and supervised the project. S.I. and C.Y.L. performed the experiments and analyzed the data. M.O. and M.K. wrote the manuscript. All authors discussed the contents of the manuscript and provided critical revisions on the manuscript.

Acknowledgements This work was supported by JSPS KAKENHI Grant Numbers 15H04190 and 17K19007 (to M.K.).



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Figure Legends Figure 1. Schematic illustration of motif-engineered chimeric receptors. a) Activation of the type I cytokine receptors. Ligand binding to the receptor leads to activation of JAK and subsequent tyrosine phosphorylation of the intracellular domain of the receptor. Multiple signaling molecules bind to the phosphorylated tyrosine motifs, and are phosphorylated by JAK for their activation. b) Principle of motif engineering. The deletion of the motif-containing region in the intracellular domain of a wild-type (WT) type I cytokine receptor results in a no-motif receptor (no motif). The addition of a tyrosine motif which can specifically recruit a signaling molecule of interest (e.g. STAT5) leads to a motif-engineered receptor. c) Activation of bottom-up engineered chimeric receptors. The extracellular domain of c-Mpl is replaced by anti-fluorescein (FL) singlechain Fv and the D2 domain of erythropoietin receptor (EpoR), whereas the intracellular motifcontaining region of c-Mpl is replaced by tyrosine motifs and linkers. The addition of FLconjugated BSA (BSA-FL) leads to oligomerization and activation of the chimeric receptor. d) The illustration of selective ablation of one of the tyrosine motifs in the bottom-up engineered receptor. The tyrosine-to-phenylalanine mutation (x) disables phosphorylation of the motif, resulting in no activation of the target signaling molecule (e.g. Shc).

Figure 2. Functional analyses of bottom-up engineered chimeric receptors. a) Illustration of the vector map and chimeric receptors. b) and c) Signal transduction analyses in Ba/F3 transductants. Parental Ba/F3 (-) and the transduced cells were unstimulated (-) or stimulated with 1 µg/ml BSA-FL (+) or 1 ng/ml IL-3. Western blotting was conducted for detecting b) phosphorylated signaling molecules and c) whole molecules. The β-tubulin blot was the loading control. d) The experimental scheme of the HSC proliferation assay. CD34-KSL, CD34-/lowcKit+Sca-1+Lineage marker- cells. e) The results of the HSC proliferation assay. EGFP-positive transduced cells including empty vector-transduced cells (-) were inoculated into 96-well plates at 50 cells/well in the presence of the indicated ligands. Viable cell counts after the 5-day culture are 15 ACS Paragon Plus Environment


plotted as mean + SE (n=6).

Figure 3. Functional analyses of top-down engineered chimeric receptors. a) Illustration of the chimeric receptors. b), c), d), and e) Signal transduction analyses in Ba/F3 transductants. Parental Ba/F3 (-) and the transduced cells were unstimulated (-) or stimulated with 1 µg/ml BSA-FL (+) or 1 ng/ml IL-3. Western blotting was conducted for detecting phosphorylated signaling molecules for b) 5S3 chimera variants and c) S35 chimera variants, and whole molecules for d) 5S3 chimera variants and e) S35 chimera variants. The β-tubulin blot was the loading control. f) The results of the HSC proliferation assay. The experimental scheme was the same as described in Figure 2d. EGFP-positive transduced cells were inoculated into 96-well plates at 50 cells/well in the presence of the indicated ligands. Viable cell counts after the 5-day culture are plotted as mean + SE (n=6).


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(a)

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(b)

Ligand

JAK

JAK

Type I cytokine receptor

P

JAK

JAK P

P

P P

P P

P

STAT5 STAT3 Shc STAT SHP2 PI3K

Activation of multiple signaling molecules

P

JAK-binding region

STAT5

P Motif-containing P region

WT

P

no motif

Motif-engineered receptor

P

(c)

(d) BSA-FL

BSA-FL

Anti-FL scFv EpoR D2 TM & JAK-binding domains of c-Mpl

Motifs & linkers

JAK

JAK

JAK STAT5 Shc STAT STAT3 3

P P P

P

P

P

P

P

P

P

JAK P STAT5 Shc STAT STAT3 3

P

JAK

JAK

P

JAK STAT5

P

P

JAK P STAT5

P

P

STAT3

P

P

STAT3

P

P

P P

Figure 1. Schematic illustration of motif-engineered chimeric receptors. a) Activation of the type I cytokine receptors. Ligand binding to the receptor leads to activation of JAK and subsequent tyrosine phosphorylation of the intracellular domain of the receptor. Multiple signaling molecules bind to the phosphorylated tyrosine motifs, and are phosphorylated by JAK for their activation. b) Principle of motif engineering. The deletion of the motif-containing region in the intracellular domain of a wild-type (WT) type I cytokine receptor results in a no-motif receptor (no motif). The addition of a tyrosine motif which can specifically recruit a signaling molecule of interest (e.g. STAT5) leads to a motif-engineered receptor. c) Activation of bottom-up engineered chimeric receptors. The extracellular domain of c-Mpl is replaced by anti-fluorescein (FL) single-chain Fv and the D2 domain of erythropoietin receptor (EpoR), whereas the intracellular motif-containing region of cMpl is replaced by tyrosine motifs and linkers. The addition of FL-conjugated BSA (BSA-FL) leads to oligomerization and activation of the chimeric receptor. d) The illustration of selective ablation of one of the tyrosine motifs in the bottom-up engineered receptor. The tyrosine-to-phenylalanine mutation (x) disables phosphorylation of the motif, resulting in no activation of the target signaling molecule (e.g. Shc). ACS Paragon Plus Environment


(a)

Anti-FL EpoR TM & D2 JBD scFv

Y+

WT

no motif

LTR

Motifs & linkers

IRES

PuroR

5

T2A

EGFP

(PCMV)

S3

5S3

S35 Anti-FL scFv EpoR D2

JAK

JAK

JAK

JAK

JAK STAT5

c-Mpl intracellular domain

(b)

(-) －

＋

IL-3

BSA-FL stimulation

WT

no motif

5

－＋－＋－

S3

JAK STAT5

5S3

JAK Shc STAT3

＋－＋－＋－

JAK STAT5 Shc STAT3

JAK Shc STAT3

Day 0

(e)

p-Shc

STAT3 STAT5 Shc

－

＋

－＋－＋－

5

S3

5S3

S35

＋－＋－＋－

Motifs & linkers

Day 9

＋

Ligand stimulation

sorting

Cell counting

60000

Cell count [cells/well]

BSA-FL stimulation

WT

TM & JAK-binding domains of c-Mpl

Day 4

Gene transduction

50000

(-)

JAK Shc STAT3 STAT5

＋

p-STAT5

(c)

JAK Shc STAT3 STAT5

Day 1

KSL

HSC purification

no motif


(d)

S35

p-STAT3

IL-3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

LTR (MoMLV)

Page 18 of 20

SCF

SCF+BSA-FL

40000

SCF+TPO

30000 20000 10000 0

(-)

β-tubulin

WT

no

5

S3

5S3

S35

motif

Figure 2. Functional analyses of bottom-up engineered chimeric receptors. a) Illustration of the vector map and chimeric receptors. b) and c) Signal transduction analyses in Ba/F3 transductants. Parental Ba/F3 (-) and the transduced cells were unstimulated (-) or stimulated with 1 μg/ml BSA-FL (+) or 1 ng/ml IL-3. Western blotting was conducted for detecting b) phosphorylated signaling molecules and c) whole molecules. The β-tubulin blot was the loading control. d) The experimental scheme of the HSC proliferation assay. CD34-KSL, CD34-/lowc-Kit+Sca-1+Lineage marker- cells. e) The results of the HSC proliferation assay. EGFP-positive transduced cells including empty vector-transduced cells (-) were inoculated into 96-well plates at 50 cells/well in the presence of the indicated ligands. Viable cell counts after the 5-day culture are plotted as mean + SE (n=6). ACS Paragon Plus Environment

Page 19 of 20


(a) 5S3 5YF

S35

SYF

3YF

5YF

SYF

3YF Anti-FL scFv EpoR D2

(b)

Shc STAT STAT3 3 5S3 (5YF)

BSA-FL stimulation －

＋

IL-3

(-)

－

JAK STAT5

STAT3

STAT3

5S3 (SYF)

＋－

5S3 (3YF)

＋－＋

5S3

－

＋

JAK STAT5 Shc

(c) BSA-FL stimulation

p-STAT3

p-STAT5

p-STAT5

p-Shc

p-Shc 5S3 (5YF)

(-)

BSA-FL stimulation

－

＋

－

5S3 (SYF)

5S3 (3YF)

＋－＋－

＋

5S3

－＋

－

＋

(e)

(-)

BSA-FL stimulation －

＋

STAT3

STAT3

STAT5

STAT5

Shc

Shc

β-tubulin

β-tubulin

JAK Shc STAT3

S35 (5YF)

(-)

p-STAT3

(d)

JAK STAT5 Shc STAT

－

S35 (3YF)

＋－＋－

S35 (5YF)

－

S35 (SYF)

JAK Shc STAT3

S35 (SYF)

＋

S35 (3YF)

＋－＋－

＋

S35

JAK

－＋

STAT3 STAT5

JAK Shc

JAK Shc

STAT5

STAT5

TM & JAK-binding domains of c-Mpl Motifs & linkers

(f)

－＋

S35

JAK

STAT3 STAT5

60000

Cell count [cells/well]

Shc STAT3

JAK STAT5

IL-3

JAK

IL-3

JAK

IL-3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

50000

SCF SCF+BSA-FL SCF+TPO

40000

30000 20000 10000 0

Figure 3. Functional analyses of top-down engineered chimeric receptors. a) Illustration of the chimeric receptors. b), c), d), and e) Signal transduction analyses in Ba/F3 transductants. Parental Ba/F3 (-) and the transduced cells were unstimulated (-) or stimulated with 1 μg/ml BSA-FL (+) or 1 ng/ml IL-3. Western blotting was conducted for detecting phosphorylated signaling molecules for b) 5S3 chimera variants and c) S35 chimera variants, and whole molecules for d) 5S3 chimera variants and e) S35 chimera variants. The βtubulin blot was the loading control. f) The results of the HSC proliferation assay. The experimental scheme was the same as described in Figure 2d. EGFP-positive transduced cells were inoculated into 96-well plates at 50 cells/well in the presence of the indicated ligands. Viable cell counts after the 5day culture are plotted as mean + SE (n=6). ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Page 20 of 20

Designing Motif-engineered receptors Antigen

Antigen

scFv


P P P P

P

P

P

P

P

P

JAK P STAT5 Shc STAT STAT3 3

scFv

P

JAK STAT5

P

P

STAT3

P

P

JAK P STAT5

P

P

STAT3

P

P P P P

Bottom-up approach

Top-down approach

ACS Paragon Plus Environment

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