Dynamics of corticosteroid receptor in neural cells

肾上腺皮质类固醇（人类的皮质醇或啮齿动物的皮质酮）在中枢神经系统中发挥多种作用，调节应激反应，情绪，学习和记忆以及各种神经内分泌功能。皮质酮（Corticosterone，CORT）在脑内的作用是由糖皮质激素受体（glucocorticoid receptor，GR）和盐皮质激素受体（mineralocorticoid receptor，MR）介导的，它们在脑内高度共定位。这些受体主要存在于细胞质中而没有配体，并且在配体结合后易位到细胞核中以充当转录因子。因此，它们的亚细胞定位是其生物活性的重要组成部分。鉴于MR和GR在中枢神经系统中的不同作用，重要的是要阐明这些受体在细胞质和细胞核之间的运输以及它们的相互作用如何受到配体或其他分子的调节以发挥转录活性。小于20-40 kDa的分子可以被动地通过核孔复合物（NPC）从细胞质扩散到细胞核，而大于40 kDa的分子则通过NPC的门控通道通过主动机制转运。输入蛋白a和输入蛋白β是跨NPC的核输入途径中用于核转运蛋白介导的底物结合的对接蛋白。本研究首先利用荧光共振能量转移技术研究了GR、MR和importins在活体培养的海马神经元和COS-1细胞中的转运，以及它们与importin α的相互作用。我们还研究了importin α亚型，importin α 1，2和3在活细胞中的动力学。接下来，我们制备了与GR结合但不能将GR转运到细胞核的importin α s的显性负性形式，并发现了一种参与皮质类固醇受体和importin α复合物的核转运的假定马达蛋白。

Adrenal corticosteroids (cortisol in humans or corticosterone in rodents) exert numerous effects in the central nervous system that regulate the stress response, mood, learning and memory, and various neuroendocrine functions. Corticosterone (CORT) actions in the brain are mediated by two corticosteroid receptors, glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), and they show a high degree of colocalization in the hippocamal region. These receptors are predominantly resided in the cytoplasm without ligand and translocated into the nucleus upon ligand binding to act as transcriptional factors. Thus their subcellualr localizations are important component of their biological activity. Given the differential action of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between cytoplasm and nucleus and their interaction are regulated by ligand or other molecules to exert transcriptional activity. Molecules less than 20-40 kDa can passively diffuse through the nuclear pore complex (NPC) from the cytoplasm to the nucleus, whereas those more than 40 kDa are transported through gated-channels of the NPC by active mechanisms. Importin a and importin β are docking proteins for karyopherin-mediated binding of substrate in a nuclear import pathway across the NPC. In the present study, we first investigated the trafficking of GR and MR, and importins in living cultured hippocampal neurons and COS-1 cells with GFP color variants, and the interaction of these receptors and importin α using fluorescent resonance energy transfer technique. We also examined dynamics of importin α subtypes, importin α 1, 2, and 3 in living cells. Next, we made dominant-negative forms of importin α s, which bind to GR, but cannot transport GR to the nucleus, and find out a putative motor protein involved in the nuclear transport of a complex of corticosteroid receptor and importin α.