Lelouvier Benjamin, Tamagno Gianluca, Kaindl Angela M, Roland Alexandre, Lelievre Vincent, Le Verche Virginia, Loudes Catherine, Gressens Pierre, Faivre-Baumann Annie, Lenkei Zsolt, Dournaud Pascal
Institut National de la Santé et de la Recherche Médicale, Unité 676, 75019 Paris, France.
J Neurosci. 2008 Apr 23;28(17):4336-49. doi: 10.1523/JNEUROSCI.4379-07.2008.
Despite the large number of G-protein-coupled receptor (GPCR) types expressed in the CNS, little is known about their dynamics in neuronal cells. Dynamic properties of the somatostatin type 2A receptor were therefore examined in resting conditions and after agonist activation in living hippocampal neurons. Using fluorescence recovery after photobleaching experiments, we found that, in absence of ligand, the sst(2A) receptor is mobile and laterally and rapidly diffuse in neuronal membranes. We then observed by live-cell imaging that, after agonist activation, membrane-associated receptors induce the recruitment of beta-arrestin 1-enhanced green fluorescent protein (EGFP) and beta-arrestin 2-EGFP to the plasma membrane. In addition, beta-arrestin 1-EGFP translocate to the nucleus, suggesting that this protein could serve as a nuclear messenger for the sst(2A) receptor in neurons. Receptors are then recruited to preexisting clathrin coated pits, form clusters that internalize, fuse, and move to a perinuclear compartment that we identified as the trans-Golgi network (TGN), and recycle. Receptor cargoes are transported through a microtubule-dependent process directly from early endosomes/recycling endosomes to the TGN, bypassing the late endosomal compartment. Together, these results provide a comprehensive description of GPCR trafficking in living neurons and provide compelling evidence that GPCR cargoes can recycle through the TGN after endocytosis, a phenomenon that has not been anticipated from studies of non-neuronal cells.
尽管中枢神经系统中表达的G蛋白偶联受体（GPCR）类型众多，但对其在神经元细胞中的动态变化却知之甚少。因此，我们研究了生长抑素2A型受体在静息状态下以及激动剂激活后的动态特性。通过光漂白后荧光恢复实验，我们发现，在没有配体的情况下，sst(2A)受体具有流动性，能在神经元膜中快速横向扩散。然后，我们通过活细胞成像观察到，激动剂激活后，膜相关受体诱导β-抑制蛋白1-增强绿色荧光蛋白（EGFP）和β-抑制蛋白2-EGFP募集到质膜。此外，β-抑制蛋白1-EGFP转位至细胞核，这表明该蛋白可能作为神经元中sst(2A)受体的核信使。随后，受体被募集到预先存在的网格蛋白包被小窝，形成聚集物，这些聚集物内化、融合并移动到我们确定为反式高尔基体网络（TGN）的核周区室，并进行循环利用。受体货物通过依赖微管的过程直接从早期内体/再循环内体运输到TGN，绕过晚期内体区室。总之，这些结果全面描述了活神经元中GPCR的运输，并提供了令人信服的证据，证明GPCR货物在胞吞作用后可通过TGN进行循环利用，这一现象在非神经元细胞研究中并未被预见到。
