Wang Y, Dun S L, Huang P, Chen C, Chen Y, Unterwald E M, Dun N J, Van Bockstaele E J, Liu-Chen L-Y
Department of Pharmacology, School of Medicine, Temple University, 3420 North Broad Street, Philadelphia, PA 19140, USA.
Neuroscience. 2006 Jul 21;140(4):1265-76. doi: 10.1016/j.neuroscience.2006.03.013. Epub 2006 May 2.
We have previously demonstrated that GEC1 interacts with the kappa opioid receptor and GEC1 expression enhances cell surface expression of the receptor in Chinese hamster ovary cells. In this study, we generated an antiserum (PA629) directed against GEC1 in rabbits, characterized its specificity, and investigated distribution of GEC1 in tissues and in brain regions and spinal cord and its subcellular localization in hypothalamic neurons in the rat. Immunofluorescence staining demonstrated that PA629 recognized HA-GEC1 transfected into Chinese hamster ovary cells, but not HA-GABARAP or HA-GATE-16, although the three share high homology. Pre-incubation of PA629 with GST-GEC1, but not GST, abolished the staining. In immunoblotting, affinity-purified PA629 (PA629p) recognized GEC1, GABARAP and GATE-16. GEC1 migrated slower than GABARAP and GATE-16, with a M(r) of 16 kDa for GEC1 and M(r) of 14 kDa for GABARAP and GATE-16. Immunoblotting results showed that GEC1 level was higher in liver and brain than in lung and heart, and very low in kidney and skeletal muscle. GEC1 was present in all rat brain regions examined and spinal cord. Immunohistochemistry demonstrated that GEC1 immunoreactivity was distributed ubiquitously in the rat CNS with highly intense immunoreactivity in various brain nuclei and motor neurons of the spinal cord. Ultrastructural examination of neurons in the paraventricular nucleus of the hypothalamus showed that GEC1 was associated with endoplasmic reticulum and Golgi apparatus and distributed along plasma membranes and in cytosol. Coupled with our previous observation that GEC1 interacts with N-ethylmaleimide-sensitive factor, these findings strongly suggest that GEC1 functions in intracellular trafficking in the biosynthesis pathway and perhaps also the endocytic pathway. The widespread distribution of GEC1 suggests that GEC1 may be associated with many proteins, in addition to the kappa opioid receptor.
我们之前已经证明，GEC1与κ阿片受体相互作用，并且GEC1的表达增强了中国仓鼠卵巢细胞中该受体的细胞表面表达。在本研究中，我们在兔体内制备了一种针对GEC1的抗血清（PA629），鉴定了其特异性，并研究了GEC1在大鼠组织、脑区、脊髓中的分布及其在下丘脑神经元中的亚细胞定位。免疫荧光染色显示，PA629能识别转染到中国仓鼠卵巢细胞中的HA-GEC1，但不能识别HA-GABARAP或HA-GATE-16，尽管这三者具有高度同源性。用GST-GEC1而非GST对PA629进行预孵育可消除染色。在免疫印迹中，亲和纯化的PA629（PA629p）能识别GEC1、GABARAP和GATE-16。GEC1迁移速度比GABARAP和GATE-16慢，GEC1的相对分子质量为16 kDa，GABARAP和GATE-16的相对分子质量为14 kDa。免疫印迹结果显示，GEC1在肝脏和脑中的水平高于肺和心脏，在肾脏和骨骼肌中非常低。GEC1存在于所有检测的大鼠脑区和脊髓中。免疫组织化学表明，GEC1免疫反应性在大鼠中枢神经系统中广泛分布，在各种脑核和脊髓运动神经元中具有高强度免疫反应性。下丘脑室旁核神经元的超微结构检查显示，GEC1与内质网和高尔基体相关，并沿质膜和胞质溶胶分布。结合我们之前观察到的GEC1与N-乙基马来酰亚胺敏感因子相互作用，这些发现强烈表明GEC1在生物合成途径以及可能的内吞途径的细胞内运输中发挥作用。GEC1的广泛分布表明，除了κ阿片受体外，GEC1可能还与许多蛋白质相关。
