Rapid receptor-proximal signaling assays for FcR gamma-containing receptors.
Novel, cell-based assays, based on bioluminescence resonance energy transfer, have been developed for FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling at receptor-proximal steps. In a stable transfectant of the HEK-293 cell line expressing human FcepsilonRIalpha, FcepsilonRIbeta, and FcRgamma-GFP2 and Syk(1-265)-Rluc fusion proteins, FcepsilonRI cross-linking markedly increased BRET2 ratios, which are the ratios of GFP2 emission to Rluc emission. These ratios reflect the FcRgamma-GFP2-Syk(1-265)-Rluc interaction in living cells. The signals are specifically inhibited by the Src-family kinase inhibitor PP2. Separately, in transient transfectants expressing GPVI, FcRgamma-GFP2, and Syk(1-265)-Rluc, the GPVI-specific ligand convulxin induced a two-fold increase in the BRET2 ratio and this increase was also inhibited by PP2. Finally, a differential assay was developed which permits the measurement of FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling in the same cell. These assays provide useful methods for monitoring FcRgamma-Syk interaction in real time in living cells and may contribute to the understanding of signal regulation through FcRgamma-containing receptors.
Mukasa
Ryuta
R
New Product Research Laboratories III, Daiichi Pharmaceutical Co., Ltd., 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan. mukasull@daiichipharm.co.jp
Terada
Yasuko
Y
Shiroishi
Machiko
M
Fujiwara
Hiroyuki
H
Hayata
Kenji
K
Morishita
Kaoru
K
Ra
Chisei
C
Takashi
Tohru
T
D015153:Blotting, Western; D002460:Cell Line; D004792:Enzyme Precursors / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008163:Luminescent Measurements / Q000379:methods*; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011961:Receptors, Fc / Q000032:analysis* / Q000096:biosynthesis / Q000502:physiology*; D015398:Signal Transduction / Q000276:immunology*; D000072377:Syk Kinase; D014162:Transfection
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D010980:Platelet Membrane Glycoproteins; D011961:Receptors, Fc; C098341:platelet membrane glycoprotein VI; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase