Schulz R, Schulz K, Wehmeyer A, Murphy J
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Königinstr. 16, D-80539 München, Germany.
Brain Res. 1998 Apr 20;790(1-2):347-56. doi: 10.1016/s0006-8993(98)00114-0.
Activation of G protein-coupled receptors triggers translocation of certain proteins from cytoplasm to cell membrane located targets. One of these cytosolic proteins is phosducin (Phd) which has been described to compete with G protein-coupled receptor kinases for Gbetagamma dimers attached to the cell membrane, thereby attenuating desensitization of activated receptors. These features of protein redistribution prompted us to examine whether stimulation of membrane associated E-prostaglandin receptors coupled to Gs causes Phd to migrate towards the plasma membrane. We made use of enhanced green fluorescence protein (EGFP), a reporter protein, to follow redistribution of Phd both by means of confocal microscopy and biochemical techniques in living neuronal NG 108-15 hybrid cells challenged with prostaglandin E1 (PGE1). The cells were transiently transfected to express Phd fused to the C-terminus of EGFP, or to express EGFP only. Overexpression of the proteins is implied by FACS analysis as well as by western blot technique, and the functional integrity of EGFP-tagged Phd was confirmed by its ability to elevate cAMP accumulation. Time-lapse imaging of single living cells by means of confocal microscopy revealed that exposure to prostaglandin causes EGFP/Phd, which is evenly spread throughout the cell, to relocate towards the membrane within few minutes. Fluorescence associated with the cell nucleus displayed little rearrangement. The principle finding that prostaglandin triggers translocation of Phd from cytosol to the cell periphery was verified with membranes prepared from EGFP/Phd expressing cells. We found maximal concentrations of membrane associated fluorescent material 5 to 7 min upon prostaglandin exposure. The present study reports for living NG 108-15 hybrid cells that PGE1 stimulation causes cytosolic Phd to translocate towards the membrane, where it is believed to bind to G protein subunits such as Gbetagamma and Galphas.
G蛋白偶联受体的激活会触发某些蛋白质从细胞质向位于细胞膜的靶点发生易位。这些胞质蛋白之一是磷光视蛋白（Phd），据描述它会与G蛋白偶联受体激酶竞争附着在细胞膜上的Gβγ二聚体，从而减弱激活受体的脱敏作用。蛋白质重新分布的这些特征促使我们研究与Gs偶联的膜相关E-前列腺素受体的刺激是否会导致Phd向质膜迁移。我们利用增强型绿色荧光蛋白（EGFP），一种报告蛋白，通过共聚焦显微镜和生化技术追踪Phd在受到前列腺素E1（PGE1）刺激的活神经元NG 108 - 15杂交细胞中的重新分布情况。这些细胞被瞬时转染以表达与EGFP C末端融合的Phd，或仅表达EGFP。通过流式细胞术分析以及蛋白质印迹技术表明蛋白质过表达，并且通过其提高cAMP积累的能力证实了EGFP标记的Phd的功能完整性。通过共聚焦显微镜对单个活细胞进行延时成像显示，暴露于前列腺素会导致在整个细胞中均匀分布的EGFP/Phd在几分钟内重新定位到膜上。与细胞核相关的荧光显示几乎没有重排。前列腺素触发Phd从胞质溶胶向细胞周边易位这一主要发现，在用表达EGFP/Phd的细胞制备的膜上得到了验证。我们发现前列腺素暴露后5至7分钟，膜相关荧光物质的浓度达到最大值。本研究报道了对于活的NG 108 - 15杂交细胞，PGE1刺激会导致胞质Phd向膜易位，据信它会在膜上与G蛋白亚基如Gβγ和Gαs结合。
