Alternative Title蛍光相互相関分光法を用いた生細胞におけるグルココルチコイド受容体2量体化機構の研究
Degree grantor/typeHokkaido University（北海道大学）
Note (General)All biomolecules are involved in some type of interaction as they perform their functions, and quantification of dissociation constants (Kds) is an important tool for understanding biomolecular interactions. One method that has great potential for determining dissociation constants is fluorescence cross-correlation spectroscopy (FCCS). FCCS uses a femtoliter-sized observation volume to observe the interaction of biomolecules labeled with different colors. The concentrations of the fluorescently labeled proteins were calculated from the autocorrelation functions in the FCCS analysis.
Human glucocorticoid receptor α (hGRα) is a well-known ligand dependent transcriptional regulatory protein. The classical view is that unliganded GRα is predominantly localize in cytoplasm, upon ligand binding the GRα is translocated to the nucleus, and then associate with the glucocorticoid response element (GRE) to transactivate the specific gene. Moreover, the GRα could transrepress the specific gene binding with GRE as a monomer. Although the dimerization of GRα is strongly related to its transcriptional activity, it is still puzzle whether GRα forms dimer in the cytoplasm or nucleus before and after DNA binding in the nucleus in living cell, and what the function of dimer formation in the cytoplasm is.
Chapter 2 describes comparative analysis of cross-correlation of fluorescent fusion proteins mCherry-EGFP and mCherry2-EGFP for selecting the appropriate pair in FCCS analysis. I examined the cross-correlation of mCherry-EGFP and mCherry2-EGFP in living cell. The cross-correlation amplitude, count per molecule and relative cross amplitude (RCA) was higher in mCherry2-EGFP compare to mCherry-EGFP. This study indicated that tandem dimer of mCherry (mCherry2) was advantageous compare to monomeric mCherry in FCCS analysis.
Members of the Rel/NFκB family of transcription factors play a vital role in the regulation of rapid cellular responses. Members of this family of proteins form homo- and heterodimers with differing affinities for dimerization. Among the most abundant and best understood of these dimers are the p50/p65 heterodimer and the homodimers of p50 or p65. The binding affinity of p50/p65 is thought to be strong as nanomolar range. However, the quantitative value of affinity, namely the Kd, for the NFκB dimers in living cells are not known yet. Chapter 3 describes quantification of the heterodimerization of p50/p65 and homodimerization of p50 and p65, using two-laser beam FCCS. For that purpose, p50 and p65 were labeled with either mCherry2 or EGFP fluorescent protein (p50-mCherry2 and p50 EGFP or p65-mCherry2 and p65-EGFP). The Kd of p50/p65 heterodimer, p50/p50 and p65/p65 homodimers were determined to be 0.46 μM, 1.78 μM and 2.59 μM respectively in the cytoplasm. The Kd of p50/p65 heterodimer, p50/p50 and p65/p65 homodimers were determined to be 1.06 μM, 1.44 μM and 1.52 μM respectively in the nucleus. These findings implicated different binding affinities of p50/p65 heterodimer, p50/p50 and p65/p65 homodimers both in the cytoplasm and nucleus and different complex formation in each region. In the present study I used p50/p50 homodimer as a positive control.
In chapter 4, the dimerization process of GRα was evaluated by two-laser-beam FCCS measurement of transiently expressed mCherry tendem dimer (mCherry2) and EGFP fused GRα in living cell. Positive cross-correlation was obtained in wild type GRα in the presence of Dex. The Kd (dissociation constant) value of mCherry2 and EGFP fused wild type GRα was determined to be 4.42 μM in the presence of Dex. On the other hand, the Kd of the DNA binding deficiency (C421G) and dimerization deficiency (A458T) mutants were 4.88 μM and 9.91 μM in the nucleus of living cell, respectively. As a result, we confirmed that DNA binding is not necessary for GRα dimerization. In addition, the effect of antagonist RU486 in the process of dimerization of GRα was also examined. Some positive cross- correlation was observed in wild type GRα in the presence of RU486. This result suggested that RU486 provoked less dimer formation of GRα and worked as partial agonist. In order to understand cytoplasmic dimerization I determined the Kd value for mCherry2 and EGFP fused ΔNLS (nuclear localization signal) and A458T-ΔNLS mutants in living cell and the estimated Kd was 3.24 μM and 6.84 μM, respectively. This finding suggested the cytoplasmic dimerization of GRα. However, the micromolar range of Kd value is higher than the expected nanomolar range, highly validate that the GRα initially shows partial dimerization in cytoplasm and later it translocate to nucleus as a monomer and it may further dimerize inside the nucleus. The diffusion properties of GRα and mutants in the nucleus and cytoplasm in the presence and absence of Dex were also compared using the distribution of diffusion constants. These findings support the dynamic monomer pathway and regulation of GRα in cytoplasm and nucleus.
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Hokkaido University（北海道大学）. 博士(生命科学)
Collection (particular)国立国会図書館デジタルコレクション > デジタル化資料 > 博士論文
Date Accepted (W3CDTF)2015-02-03T05:25:05+09:00
Data Provider (Database)国立国会図書館 : 国立国会図書館デジタルコレクション