An integrated-molecule-format multicolor probe for monitoring multiple activities of a bioactive small molecule.
Bioactive small molecules, including steroids, activate multiple signaling pathways in mammalian cells. However, current technologies cannot illuminate such multiple effects of a ligand in mammalian cells. Here, we demonstrate integrated-molecule-format multicolor systems simultaneously visualizing bifacial activities of a ligand, where estrogen receptor alpha (ERalpha) was exemplified to demonstrate the present technology. First, we developed a single-molecule-format probe emitting red bioluminescence for imaging interaction between the phosphorylated ligand binding domain of ERalpha (ER LBD) and the Src homology-2 (SH2) domain of Src. The SH2 domain-linked ER LBD was sandwiched between dissected N- and C-terminal fragments of Pyrophorus plagiophthalamus (click beetle) luciferase emitting red bioluminescence. Second, another single-molecule-format bio-luminescent probe emitting green bioluminescence was constructed to visualize intramolecular interaction between ER LBD and LXXLL motifs. Mammalian cells carrying the two probes emit red and/or green light in response to agonistic and antagonistic activities of a ligand, which correspond to its genomic and nongenomic activities, respectively. Third, the two probes were assembled to make an single-molecule-format multicolor indicator, in which all of the components for ligand sensing and multiple-light emission were integrated. The probe emitted characteristic light spectra in response to various agonists and antagonists. This is the first example where (i) protein phosphorylation was recognized with a single bioluminescent probe and (ii) bifacial activities of a ligand, either agonistic or antagonistic, were simultaneously visualized with multiple colors.
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba 305-8569, Japan.
Sung Bae
SB
Kim
Yoshio
Y
Umezawa
Kira A
KA
Kanno
Hiroaki
H
Tao
D000818:Animals; D001665:Binding Sites; D002477:Cells / Q000166:cytology / Q000378:metabolism; D001517:Coleoptera / Q000201:enzymology; D047628:Estrogen Receptor alpha / Q000032:analysis / Q000378:metabolism; D008024:Ligands; D008156:Luciferases / Q000737:chemistry*; D049408:Luminescent Agents / Q000737:chemistry*; D008163:Luminescent Measurements / Q000379:methods*; D010766:Phosphorylation; D012680:Sensitivity and Specificity; D018909:src Homology Domains / Q000502:physiology*; D019061:src-Family Kinases / Q000032:analysis / Q000378:metabolism
D047628:Estrogen Receptor alpha; D008024:Ligands; D049408:Luminescent Agents; D008156:Luciferases; D019061:src-Family Kinases