Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors
Constitutive activation drives compartment-selective endocytosis and axonal targeting of type 1 cannabinoid receptors
复制标题
DOI：
10.1523/jneurosci.5437-05.2006
复制
发表时间：
2006-03-22
影响因子：
5.3
通讯作者：
Lenkei, Z
中科院分区：
文献类型：
--
作者：
Leterrier, C;Lainé, J;Lenkei, Z
1型大麻素受体（CB 1 R）是脑中最丰富的G蛋白偶联受体（GPCR）之一，主要定位于GABA能神经元的轴突。与其他几种神经元GPCR一样，CB 1 R显示出显著的体外组成型活性（即，在没有配体的情况下自发活化）。然而，组成型GPCR活性的明确生物学作用仍然缺乏。这个问题是通过研究的内源性或转染CB 1 Rs在培养的海马神经元，使用光学和电子显微镜的细胞内贩运的组成性激活的后果。我们发现，组成性活动的结果在一个永久的内吞和循环，这是有限的体树突区室。因此，CB 1 Rs通过胞吞作用从体树突隔室中的质膜连续去除，但不在轴突中，CB 1 Rs在轴突表面上积累。通过与反向激动剂1-（2，4-二氯苯基）-5-（4-碘苯基）-4-甲基N- 4-吗啉基-1H-吡唑-3-甲酰胺（AM 281）短期孵育来阻断组成型活性导致再循环的CB 1 R在体树突质膜上的螯合。通过共转染显性阴性蛋白质长期抑制内吞作用导致表面结合CB 1 Rs的轴突极化受损。动力学分析表明，大多数新合成的CB 1 R首先到达体树细胞质膜，在被递送到轴突之前，它们通过AM 281敏感性组成性内吞作用迅速被去除。因此，组成型活性驱动的体树突细胞内吞作用是CB 1 R的适当轴突靶向所必需的，代表了轴突GPCR的一种新的构象依赖性靶向机制。
The type 1 cannabinoid receptor (CB1R) is one of the most abundant G-protein-coupled receptors (GPCRs) in the brain, predominantly localized to axons of GABAergic neurons. Like several other neuronal GPCRs, CB1R displays significant in vitro constitutive activity (i.e., spontaneous activation in the absence of ligand). However, a clear biological role for constitutive GPCR activity is still lacking. This question was addressed by studying the consequences of constitutive activation on the intracellular trafficking of endogenous or transfected CB1Rs in cultured hippocampal neurons using optical and electron microscopy. We found that constitutive activity results in a permanent cycle of endocytosis and recycling, which is restricted to the somatodendritic compartment. Thus, CB1Rs are continuously removed by endocytosis from the plasma membrane in the somatodendritic compartment but not in axons, where CB1Rs accumulate on surface. Blocking constitutive activity by short-term incubation with inverse agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methylN- 4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) results in sequestration of recycled CB1Rs on the somatodendritic plasma membrane. Long-term inhibition of endocytosis by cotransfection of dominant-negative proteins results in impaired axonal polarization of surface-bound CB1Rs. Kinetic analysis shows that the majority of newly synthesized CB1Rs arrive first to the somatodendritic plasma membrane, from where they are rapidly removed by AM281-sensitive constitutive endocytosis before being delivered to axons. Thus, constitutive-activity driven somatodendritic endocytosis is required for the proper axonal targeting of CB1R, representing a novel, conformation-dependent targeting mechanism for axonal GPCRs.