Dunah A W, Yasuda R P, Wang Y H, Luo J, Dávila-García M, Gbadegesin M, Vicini S, Wolfe B B
Department of Pharmacology, Georgetown University School of Medicine, Washington, D.C. 20007, USA.
J Neurochem. 1996 Dec;67(6):2335-45. doi: 10.1046/j.1471-4159.1996.67062335.x.
A polyclonal antibody for the NMDA receptor subunit NR2D has been developed that identifies an approximately 160-kDa band on immunoblots from NR2D transfected cells and CNS tissues. No cross-reactivity is seen with other NMDA receptor subunits. The NR2D receptor subunit is N-glycosylated in both brain and transfected cells. Transfected cells expressing NR2D are immunofluorescently labeled, whereas untransfected cells or cells transfected with other NMDA receptor subunit cDNAs are not. Similarly, the NR2D subunit is selectively and quantitatively immunoprecipitated, whereas the NR1, NR2A, or NR2B subunit is not. The relative densities of the NR2D subunit in nine areas of postnatal day 7 and adult rat brains have been determined by quantitative immunoblotting. NR2D was expressed at highest levels in the thalamus, midbrain, medulla, and spinal cord, whereas intermediate levels of this subunit were found in the cortex and hippocampus. Low or undetectable levels were seen in the olfactory bulb, striatum, and cerebellum. Following a peak after the first week of birth, NR2D protein levels decreased by about twofold in adulthood in all rat brain regions examined. More complete ontogenic profiles were determined for the diencephalon, telencephalon, and spinal cord where similar ontogenic patterns were seen. NR2D protein is present at high levels at embryonic stages of development, rises to a peak at postnatal day 7, and decreases but remains measurable during late postnatal life. This study demonstrates the generation and characterization of an antibody selective for the NR2D NMDA receptor subunit as well as a determination of the distribution and ontogenic profile of this subunit in rat brain. The results suggest that native NMDA receptors containing the NR2D subunit may have functional roles not only in the young brain but also in adult brain.
已开发出一种针对NMDA受体亚基NR2D的多克隆抗体，该抗体在来自NR2D转染细胞和中枢神经系统组织的免疫印迹上可识别出一条约160 kDa的条带。未观察到与其他NMDA受体亚基的交叉反应。NR2D受体亚基在脑和转染细胞中均进行了N-糖基化。表达NR2D的转染细胞被免疫荧光标记，而未转染的细胞或用其他NMDA受体亚基cDNA转染的细胞则未被标记。同样，NR2D亚基被选择性地和定量地免疫沉淀，而NR1、NR2A或NR2B亚基则未被沉淀。通过定量免疫印迹法测定了出生后第7天和成年大鼠脑九个区域中NR2D亚基的相对密度。NR2D在丘脑、中脑、延髓和脊髓中表达水平最高，而在皮质和海马中发现该亚基的表达水平中等。在嗅球、纹状体和小脑中观察到低水平或无法检测到的水平。在出生后的第一周达到峰值后，在所有检测的大鼠脑区域中，NR2D蛋白水平在成年期下降了约两倍。对间脑、端脑和脊髓确定了更完整的个体发育图谱，观察到类似的个体发育模式。NR2D蛋白在胚胎发育阶段含量很高，在出生后第7天升至峰值，在出生后晚期下降但仍可检测到。本研究证明了针对NR2D NMDA受体亚基的抗体的产生和特性，以及该亚基在大鼠脑中的分布和个体发育图谱的确定。结果表明，含有NR2D亚基的天然NMDA受体可能不仅在幼脑中而且在成脑中都具有功能作用。
