Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Mar 15;64(6):2113-9.
doi: 10.1158/0008-5472.can-03-2972.

Molecular imaging of drug-modulated protein-protein interactions in living subjects

Ramasamy Paulmurugan  1 , Tarik F Massoud, Jing Huang, Sanjiv S Gambhir
Affiliations

Molecular imaging of drug-modulated protein-protein interactions in living subjects

Ramasamy Paulmurugan et al. Cancer Res. .

Abstract

Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor kappaB promoter/enhancer elements using tumor necrosis factor alpha, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Schematic diagram of rapamycin-mediated synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation strategy. In this strategy, N-terminal and COOH-terminal portions of hRLUC fragments are attached to proteins X and Y, respectively, through a short peptide linker GGGGSGGGGS. The N and C portions of hRLUC fragments are closely approximated by the dimerization of proteins FRB and FKBP12 only in the presence of the small molecule rapamycin, and this, in turn, leads to recovered activity of the hRLUC protein.
Fig. 2
Fig. 2
Schematic representation of the plasmid constructs made and used in this study. Shown are the components of the genes (Nhrluc, N portion of synthetic renilla luciferase fragment; Chrluc, C portion of synthetic renilla luciferase fragment; FRB and FKBP12 are the rapamycin-binding human proteins) and promoters [CMV, cytomegalovirus early promoter/enhancer elements; nuclear factor κB (NFκB), tumor necrosis factor promoter sequences]. (G4S)2 is the amino acid sequence of the linkers used between the reporter fragments and the interacting proteins. A, pCMV-Nhrluc-FRB. B, pNFκB-Nhrluc-FRB. C, pCMV-FKBP12-Chrluc. D, pCMV-Nhrluc. E, pCMV-Chrluc.
Fig. 3
Fig. 3
Flow chart showing the different experimental strategies used in this study.
Fig. 4
Fig. 4
Larger graph, rapamycin-mediated complementation of synthetic renilla lucif-erase (hRLUC) activity in transiently transfected 293T cells. The 293T cells were transiently transfected with plasmid constructs Nhrluc-FRB (N) or Nhrluc-FRB plus FKBP12-Chrluc (N+C), either with rapamycin [N(+RAP) or N+C(+RAP)] or without rapamycin [N(−RAP) or N+C(−RAP)]. The cells cotransfected with Nhrluc-FRB plus FKBP12-Chrluc that received rapamycin showed significant recovered hRLUC signal. The error bars represent the SE for triplicate determinations. Inset graph, rapamycin was added to the lysates collected from the cells cotransfected with Nhrluc-FRB plus FKBP12-Chrluc to modulate extracellular dimerization of FRB and FKBP12. The cell lysates receiving rapamycin (Rap) showed significant dimerization-associated recovery of hRLUC complementation signal. The error bars represent the SE for triplicate determinations.
Fig. 5
Fig. 5
A, graph showing the results obtained from the cells cotransfected with Nhrluc-FRB and FKBP12-Chrluc on addition of different concentrations of rapamycin (0.0195–40 nM). The optimal concentration of rapamycin for efficient dimerization-associated recovery of complemented synthetic renilla luciferase (hRLUC) activity is >10 nM. Saturation of this activity is seen at 10 nM rapamycin. The error bars represent the SE for triplicate determinations. B, the cells cotransfected with Nhrluc-FRB and FKBP12-Chrluc at a fixed concentration of rapamycin (20 nM) subsequently received different concentrations of ascomycin to demonstrate the competitive binding of these two compounds for FKBP12 and the associated reduction in the complemented hRLUC activity. The error bars represent the SE for triplicate determinations.
Fig. 6
Fig. 6
Graph showing the variable extents of synthetic renilla luciferase (hRLUC) complementation, modulated by rapamycin, obtained in the different cell lines (293T, U87, N2a, and HeLa) studied. Cells were cotransfected with Nhrluc-FRB and FKBP12- Chrluc exposed to 20-nM concentration of rapamycin and assayed after 24 h. The results show the highest signal from 293T, followed by N2a, U87, and HeLa cells. The error bars represent the SE for triplicate determinations.
Fig. 7
Fig. 7
Graph showing the results obtained from 293T cells cotransfected with Nhrluc-FRB and FKBP12-Chrluc and dually modulated at the expression level [by controlling synthetic renilla luciferase (hRLUC) transcription with varying concentrations of tumor necrosis factor α (TNF-α)] and at the dimerization level (by controlling the rapamycin concentration). The cells cotransfected with constructs driven by the CMV promoter and receiving rapamycin [(Rap+/TNF-α−) and (Rap+/TNF-α+)] showed significant increase in the complemented hRLUC signal. The cells cotransfected with constructs driven by the nuclear factor κB promoter showed significant signal only when receiving TNF-α and rapamycin (Rap+/TNF-α+). The error bars represent the SE for triplicate determinations.
Fig. 8
Fig. 8
Optical charged coupled device imaging of living mice implanted i.p. with transiently cotransfected 293T cells to study the concentrations of rapamycin required for efficient heterodimerization of FRB and FKBP12 by using the synthetic renilla luciferase protein fragment-assisted complementation strategy. At 24 h after cell implantation, the group of animals receiving 10 μg rapamycin emitted a signal (2.38 × 103 p/s/cm2/sr) that was similar to mock transfection levels. The group of animals that received 25 μg and 50 μg rapamycin emitted signals of 6.0 × 103 p/s/cm2/sr and 1.2 × 104 p/s/cm2/sr, respectively. See inset graphical display of these signals; the error bars are the SE for three mice.
Fig. 9
Fig. 9
Optical charged coupled device imaging of living mice carrying i.v. injected 293T cells transiently cotransfected with Nhrluc-FRB and FKBP12-Chrluc. The animals not receiving rapamycin showed only a mean background signal of 4 ± 1 × 103 p/s/cm2/sr at all of the time points studied. The animals receiving repeated injections of rapamycin emitted signals, originating from the region of the liver, that were threefold (mean, 1.6 × 104 p/s/cm2/sr) and fivefold (mean, 3.0 × 104 p/s/cm2/sr) higher than background (P < 0.05) at 24 h and 48 h after the injection of rapamycin, respectively. (R−, animals not receiving rapamycin; R+, animals receiving rapamycin).
Fig. 10
Fig. 10
Optical charged coupled device imaging of living mice carrying s.c. injected 293T cells transiently cotransfected with Nhrluc-FRB and FKBP12-Chrluc. The cells were pre-exposed in cell culture to 20 nM rapamycin for 24 h after cotransfection, and 5 × 106 of these cells then were implanted s.c. The groups of animals were imaged immediately (time 0) and 24 h, 48 h, and 96 h after injecting repeated doses of 50 μg rapamycin. The results showed significant increase in complemented synthetic renilla luciferase signal only from the group receiving repeated doses of rapamycin (R+, animals receiving rapamycin; R−, animals not receiving rapamycin). See inset graphical display of these signals; the error bars are the SE for three mice.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Michnick SW. Exploring protein interactions by interaction-induced folding of proteins from complementary peptide fragments. Curr Opin Struct Biol. 2001;11:472–7. - PubMed
    1. Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989;340:245–6. - PubMed
    1. Williams NE. Immunoprecipitation procedures. Methods Cell Biol. 2000;62:449–53. - PubMed
    1. Bollag DM. Gel-filtration chromatography. Methods Mol Biol. 1994;36:1–9. - PubMed
    1. Hansen JC, Lebowitz J, Demeler B. Analytical ultracentrifugation of complex macromolecular systems. Biochemistry. 1994;33:13155– 63. - PubMed

Publication types

MeSH terms