Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, 001-0021, Japan.
Sci Rep. 2017 Jun 28;7(1):4336. doi: 10.1038/s41598-017-04499-7.
Glucocorticoid receptor (GRα) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GRα resides in the cytoplasm, relocates to the nucleus after ligand binding, and then associates with a specific DNA sequence, namely a glucocorticoid response element (GRE), to activate a specific gene as a homodimer. It is still a puzzle, however, whether GRα forms the homodimer in the cytoplasm or in the nucleus before DNA binding or after that. To quantify the homodimerization of GRα, we constructed the spectrally different fluorescent protein tagged hGRα and applied fluorescence cross-correlation spectroscopy. First, the dissociation constant (K) of mCherry-fused hGRα or EGFP-fused hGRα was determined in vitro. Then, K of wild-type hGRα was found to be 3.00 μM in the nucleus, which was higher than that in vitro. K of a DNA-binding-deficient mutant was 3.51 μM in the nucleus. This similarity indicated that GRα homodimerization was not necessary for DNA binding but could take place on GRE by means of GRE as a scaffold. Moreover, cytoplasmic homodimerization was also observed using GRα mutated in the nuclear localization signal. These findings support the existence of a dynamic monomer pathway and regulation of GRα function both in the cytoplasm and nucleus.
糖皮质激素受体 (GRα) 是一种众所周知的配体依赖性转录调节蛋白。经典观点认为，未配体结合的 GRα 位于细胞质中，在配体结合后转移到细胞核内，然后与特定的 DNA 序列（即糖皮质激素反应元件 (GRE)）结合，作为同源二聚体激活特定基因。然而，在 DNA 结合之前或之后，GRα 是否在细胞质或细胞核中形成同源二聚体，仍然是一个谜。为了定量测定 GRα 的同源二聚化，我们构建了光谱不同的荧光蛋白标记的 hGRα，并应用荧光相关光谱技术。首先，在体外测定了 mCherry 融合 hGRα 或 EGFP 融合 hGRα 的解离常数 (K)。然后发现野生型 hGRα 的 K 在核内为 3.00 μM，高于体外值。DNA 结合缺陷突变体的 K 在核内为 3.51 μM。这种相似性表明，GRα 同源二聚化对于 DNA 结合不是必需的，但可以通过 GRE 作为支架在 GRE 上发生。此外，还观察到核定位信号突变的 GRα 细胞质同源二聚化。这些发现支持了 GRα 功能在细胞质和细胞核中存在动态单体途径和调节的存在。
