Intracellular signal transduction is regulated by a variety of transmembrane receptors. Many researchers have aimed to arbitrarily regulate the intracellular signaling and subsequent cell fate with artificial receptors, of which the ligand recognition and signaling properties could be artificially designed. Although several architectures of homodimeric artificial receptors have been reported, engineering of heterodimeric receptors, which are abundant among natural receptors, have yet to be thoroughly investigated. In this study, we rationally design artificial heterodimeric receptors for activating target signaling molecules. We locate a tyrosine motif on an engineered tyrosine kinase domain, which is further connected to a small molecule-responsive heterodimeric module, attaining a pair of heterodimeric receptors with different tyrosine motifs within the pair.
Fig: Rational design of heterodimeric designer receptors. (a) The designer receptors consist of the following 3 parts; 1) an engineered scaffold-less c-KIT which can phosphorylate the tyrosine motif, 2) a tyrosine motif, which recruits a target signaling molecule when phosphorylated, and 3) a small molecule-responsive heterodimerizing domain (e.g. AP21967-responsive FKBP/FRBT2098L complex). (b) The activation profiles of designer receptors without membrane localization. The STAT3- and STAT5-binding motifs were connected on either FKBP chimera or FRB chimera. The symbols Δ, 3, and 5 mean no motif, the STAT3-binding motif, and the STAT5-binding motif, respectively. In western blotting, parental Ba/F3 and the transduced cells were unstimulated (−) or stimulated with 50 nM AP21967 (+). Phospho-STAT3, whole STAT3, phospho-STAT5, whole STAT5, HA tag (FKBP chimera), V5 tag (FRB chimera), Myc tag (both FKBP and FRB chimeras; upper and lower bands, respectively), and GAPDH (loading control) were detected using corresponding primary antibodies. Full-length blots are presented in Supplementary Fig. 8.
The resultant heterodimeric receptors successfully activate target signaling molecules and even control cell proliferation levels according to the properties of tyrosine motifs connected. Thus, our heterodimeric receptors may open a new era of tailor-made designer receptors, which could be useful for cell therapy against intractable diseases.
Kongkrongtong, T., Zhang, R. & Kawahara, M. Rational design of heterodimeric receptors capable of activating target signaling molecules. Sci Rep 11, 16809 (2021). https://doi.org/10.1038/s41598-021-96396-3