Boudin H, Lazaroff B, Bachelet C M, Pélaprat D, Rostène W, Beaudet A
Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
J Comp Neurol. 2000 Sep 11;425(1):45-57.
Earlier studies have demonstrated overexpression of NT1 neurotensin receptors in rat brain during the first 2 weeks of life. To gain insight into this phenomenon, we investigated the identity and distribution of NT1 receptor proteins in the brain of 10-day-old rats by using two different NT1 antibodies: one (Abi3) directed against the third intracellular loop and the other (Abi4) against the C-terminus of the receptor. Immunoblot experiments that used Abi3 revealed the presence of two differentially glycosylated forms of the NT1 receptor in developing rat brain: one migrating at 54 and the other at 52 kDa. Whereas the 54-kDa form was expressed from birth to adulthood, the 52-kDa form was detected only at 10 and 15 days postnatal. Only the 52-kDa isoform was recognized by Abi4. By immunohistochemistry, both forms of the receptor were found to be predominantly expressed in cerebral cortex and dorsal hippocampus, in keeping with earlier radioligand binding and in situ hybridization data. However, whereas Abi4 immunoreactivity was mainly concentrated within nerve cell bodies and extensively colocalized with the Golgi marker alpha-mannosidase II, Abi3 immunoreactivity was predominantly located along neuronal processes. These results suggest that the transitorily expressed 52-kDa protein corresponds to an immature, incompletely glycosylated and largely intracellular form of the NT1 receptor and that the 54-kDa protein corresponds to a mature, fully glycosylated, and largely membrane-associated form. They also indicate that antibodies directed against different sequences of G-protein-coupled receptors may yield isoform-specific immunohistochemical labeling patterns in mammalian brain. Finally, the selective expression of the short form of the NT1 receptor early in development suggests that it may play a specific role in the establishment of neuronal circuitry.
早期研究表明，在出生后的前两周，大鼠脑中NT1神经降压素受体过度表达。为深入了解这一现象，我们使用两种不同的NT1抗体，研究了10日龄大鼠脑中NT1受体蛋白的特性和分布：一种（Abi3）针对第三个细胞内环，另一种（Abi4）针对受体的C末端。使用Abi3的免疫印迹实验显示，在发育中的大鼠脑中存在两种糖基化形式不同的NT1受体：一种迁移率为54 kDa，另一种为52 kDa。54 kDa的形式从出生到成年都有表达，而52 kDa的形式仅在出生后10天和15天被检测到。只有52 kDa的异构体被Abi4识别。通过免疫组织化学发现，两种形式的受体主要在大脑皮层和背侧海马中表达，这与早期放射性配体结合和原位杂交数据一致。然而，Abi4免疫反应主要集中在神经细胞体内，并与高尔基体标记物α-甘露糖苷酶II广泛共定位，而Abi3免疫反应主要位于神经元突起上。这些结果表明，短暂表达的52 kDa蛋白对应于NT1受体的一种未成熟、糖基化不完全且主要位于细胞内的形式，而54 kDa蛋白对应于一种成熟、完全糖基化且主要与膜相关的形式。它们还表明，针对G蛋白偶联受体不同序列的抗体可能在哺乳动物脑中产生异构体特异性的免疫组织化学标记模式。最后，NT1受体短形式在发育早期的选择性表达表明它可能在神经元回路的建立中发挥特定作用。
