Fluorescence resonance energy transfer reveals interleukin (IL)-1-dependent aggregation of IL-1 type I receptors that correlates with receptor activation.
Fluorescence resonance energy transfer (FRET) was used to investigate whether interleukin-1 (IL-1) causes the aggregation of IL-1 type I receptors (IL-1 RI) at the cell surface. For these experiments, a noncompetitive anti-IL1 RI monoclonal antibody, M5, was labeled separately with a donor probe, fluorescein isothiocyanate, or with an acceptor carbocyanine probe, Cy3. Donor-labeled M5 and acceptor-labeled M5 were simultaneously bound to transfected mouse IL-1 RI on either C-127 mouse mammary carcinoma cells or on Chinese hamster ovary (CHO)-K1 cells, and the ratio of acceptor emission at 590 nm to donor emission at 525 nm (excitation at 488 and 514 nm) was monitored with flow cytometry as an indicator of FRET. Addition of a saturating concentration of human IL-1 alpha at 22 degrees C causes a time-dependent increase in FRET for both cell lines that indicates IL-1-dependent self-association of IL-1 RI. Binding of the IL-1 receptor antagonist at 22 degrees C causes little or no FRET for both cell lines, indicating a correlation between receptor aggregation and the ability of the ligand to stimulate a functional response. When donor-labeled and acceptor-labeled Fab fragments of M5 are used to monitor FRET, IL-1 alpha causes efficient energy transfer in the CHO-K1 cells at 22 degrees C, but not at 4 degrees C. In contrast, IL-1 alpha causes much less FRET at 22 degrees C in C-127 cells when the M5 Fab fragments are used instead of the intact bivalent M5. In a striking parallel, IL-1 alpha-dependent activation of prostaglandin E2 production depends on the bivalent M5 antibody in the C-127 cells, but is independent of this monoclonal antibody in the CHO-K1 cells. These results provide a strong correlation between the ability of IL-1 to cause the aggregation of IL-1 RI and the stimulation of a functional response.
D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D006224:Cricetinae; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000378:metabolism* / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D051381:Rats; D017472:Receptors, Interleukin-1 / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012795:Sialoglycoproteins / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014162:Transfection
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; C507763:IL1RN protein, human; C507764:Il1rn protein, mouse; D007140:Immunoglobulin Fab Fragments; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D016650:Fluorescein-5-isothiocyanate