Functional Analysis of Tyrosinekinase in B Cells with Fluorescent Molecular Rotars

1997 Fiscal Year Final Research Report Summary

• Project/Area Number: 08672483
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: Suzuka University of Medical Science and Technology (1997); Nagoya City University (1996)
• Principal Investigator: NOJI Masahide Suzuka University of Medical Science and Technology, Faculty of Medical Nutriology, Professor, 医療栄養学科, 教授 (70080190)
• Co-Investigator(Kenkyū-buntansha): FURUNO Tadahide Nagoya City University, Faculty of Pharmaceutical Sciences, Lecturer, 薬学部, 講師 (80254308)
• Project Period (FY): 1996 – 1997
• Keywords: tyrosinekinase / phosphorylated peptides / monoclonal antibody / ITAM / Image analysis

Research Abstract

The IgE receptors (Fc epsilon RI) of rat basophilic leukemia cells (RBL-2H3) contain a consensus sequence termed immunoreceptor tyrosine-based activation motif (ITAM) in beta and gamma subunits. In ITAM there exist two phosphotyrosines, which play an important role in signal transduction cascade.
In this study, we have synthesized tyrosine phosphorylated ITAM peptides for beta and gamma subunits in rat IgE receptors, that is, (DRLYEELNHVYSPIYSELC) and (DAVYTGLNTRNQETYETLC) for beta and gamma subunits, respectively, where Y represents phosphorylated tyrosine. These phosphorylated peptides were synthesized by solid-phase Fmoc chemistry by using Fmoc-Tyr-(PO_3Me_2).
[Image Analysis of ITAM in RBL-2H3 Cells]
Among the obtained monoclonal antibodies against the peptides containing phosphorylated tyrosines, J-59 and J-69 monoclonal antibodies recognized proteins existing near cell membranes and cytoplasm after the antigen stimulation of the cells, especially the later one binds more strongly to proteins existing near cell membranes than the former. Encouraged by this result, time course of J-69 recognition for the membrane proteins after the stimulation was pursued by analyzing images obtained by a confocal fluorescence microscopy. In 5 and 15minutes after the stimulation J-69 recognized more preferentially ITAM near cell membranes. However, the fluorescence intensity decreased remarkably in 1 hr and 2 hr later, indicating the removal of antibodies from the membrane protein. This phenomenon can be explained by taking account of dephosphorylation of the gamma subunits by phosphatase with the resultant J-69 removal.