Laffray Sophie, Tan Kelly, Dulluc Josette, Bouali-Benazzouz Rabia, Calver Andrew R, Nagy Frédéric, Landry Marc
INSERM U 862, Institut François Magendie, Université Bordeaux 2, Bordeaux, France.
Eur J Neurosci. 2007 Mar;25(5):1402-16. doi: 10.1111/j.1460-9568.2007.05398.x.
Gamma-aminobutyric acid type B receptors (GABAB) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABAB1 and GABAB2 subunits, a process that occurs within the endoplasmic reticulum. However, the mechanisms that regulate GABAB receptor oligomerization at the plasma membrane remain largely unknown. We first characterized the functional cytoarchitecture of an organotypic co-culture model of rat dorsal root ganglia and spinal cord. Subsequently, we studied the interactions between GABAB subunits after chronic stimulation of sensory fibres with capsaicin. Surface labelling of recombinant proteins showed a decrease in subunit co-localization and GABAB2 labelling, after capsaicin treatment. In these conditions, fluorescence lifetime imaging measurements further demonstrated a loss of interactions between green fluorescent protein-GABAB1b and t-dimer discosoma sp red fluorescent protein-GABAB2 subunits. Finally, we established that the GABAB receptor undergoes clathrin-dependent internalization and rapid recycling to the plasma membrane following activation with baclofen, a GABAB agonist. However, in cultures chronically stimulated with capsaicin, the agonist-induced endocytosis was decreased, reflecting changes in the dimeric state of the receptor. Taken together, our results indicate that the chronic stimulation of sensory fibres can dissociate the GABAB heterodimer and alters its responsiveness to the endogenous ligand. Chronic stimulation thus modulates receptor oligomerization, providing additional levels of control of signalling.
γ-氨基丁酸B型受体（GABAB）是G蛋白偶联受体，介导大脑中的GABA能抑制作用。它们的功能表达依赖于GABAB1和GABAB2亚基之间异二聚体的形成，这一过程发生在内质网中。然而，调节质膜上GABAB受体寡聚化的机制在很大程度上仍然未知。我们首先对大鼠背根神经节和脊髓的器官型共培养模型的功能细胞结构进行了表征。随后，我们研究了用辣椒素慢性刺激感觉纤维后GABAB亚基之间的相互作用。重组蛋白的表面标记显示，辣椒素处理后亚基共定位和GABAB2标记减少。在这些条件下，荧光寿命成像测量进一步证明绿色荧光蛋白-GABAB1b和t-二聚体盘珊瑚红色荧光蛋白-GABAB2亚基之间的相互作用丧失。最后，我们确定GABAB受体在用GABAB激动剂巴氯芬激活后经历网格蛋白依赖性内化并迅速循环回到质膜。然而，在用辣椒素慢性刺激的培养物中，激动剂诱导的内吞作用减少，反映了受体二聚体状态的变化。综上所述，我们的结果表明，感觉纤维的慢性刺激可以使GABAB异二聚体解离，并改变其对内源性配体的反应性。因此，慢性刺激调节受体寡聚化，提供了额外的信号控制水平。
