Veyrat-Durebex Christelle, Pomerleau Luc, Langlois Daniel, Gaudreau Pierrette
Laboratory of Neuroendocrinology of Aging, Centre Hospitalier de l'Université de Montréal Research Center, Notre-Dame Hospital and Department of Medicine, University of Montreal, Montreal, Quebec, Canada H2L 4M1.
J Cell Physiol. 2005 May;203(2):335-44. doi: 10.1002/jcp.20233.
Internalization and intracellular trafficking of the growth hormone-releasing hormone receptor (GHRH-R) were studied in rat anterior pituitary and human (h) and rat (r) GHRH-R-transfected BHK cells, with the GHRH agonist, [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2), Ala(8), Ala(15), Lys(22)]hGHRH(1-29)NH(2) (Fluo-GHRH). Time- and temperature-dependent internalization of stimulated GHRH-R was blocked by phenyl arsine oxide (PAO) in both cell types. In anterior pituitary and rGHRH-R-transfected BHK cells, only filipin III and cerulenin blocked receptor-mediated internalization of Fluo-GHRH while in hGHRH-R-transfected BHK cells, only hyperosmolar sucrose inhibited this process. These results suggest that hGHRH-R internalization is clathrin-dependent, while fatty acid acylation of rGHRH-R appears to be a prerequisite to caveolin-dependent internalization. Experiments in anterior pituitary using Bodipy-FL-C(5) ganglioside GM1, a specific marker of lipid rafts such as caveolae, confirmed this latter pathway. Co-localization of Fluo-GHRH with LysoTracker indicated that Fluo-GHRH was directed to acidic organelles in both cell types. Finally, studies using cycloheximide and monensin showed that upon stimulation with GHRH, an optimal concentration of functional GHRH-R was maintained at the plasma membrane due to de novo synthesis and recycling in pituitary cells and to de novo synthesis solely in hGHRH-R-transfected BHK cells. This first study on the dynamics of the GHRH/GHRH-R complexes using fluorescence imaging in a native environment compared to cell system models, revealed that both receptor primary structure and concentration at the plasma membrane play important roles in internalization and trafficking of specific G-protein-coupled receptors (GPCR).
利用生长激素释放激素激动剂[N(α)-5-羧基荧光素-D-丙氨酸(2)，丙氨酸(8)，丙氨酸(15)，赖氨酸(22)]人促生长激素释放激素(1-29)NH₂(Fluo-GHRH)，在大鼠垂体前叶以及人源和大鼠源促生长激素释放激素受体(GHRH-R)转染的BHK细胞中研究了GHRH-R的内化和细胞内运输。在两种细胞类型中，苯砷氧化物(PAO)均阻断了刺激后的GHRH-R的时间和温度依赖性内化。在垂体前叶和大鼠GHRH-R转染的BHK细胞中，只有制霉菌素III和浅蓝菌素阻断了Fluo-GHRH的受体介导内化，而在人GHRH-R转染的BHK细胞中，只有高渗蔗糖抑制了这一过程。这些结果表明，人GHRH-R的内化是网格蛋白依赖性的，而大鼠GHRH-R的脂肪酸酰化似乎是小窝蛋白依赖性内化的先决条件。在垂体前叶使用Bodipy-FL-C₅神经节苷脂GM1（一种脂筏如小窝的特异性标记物）进行的实验证实了后一种途径。Fluo-GHRH与溶酶体追踪染料的共定位表明，Fluo-GHRH在两种细胞类型中均被导向酸性细胞器。最后，使用环己酰亚胺和莫能菌素的研究表明，在GHRH刺激后，由于垂体细胞中的从头合成和再循环以及仅在人GHRH-R转染的BHK细胞中的从头合成，功能性GHRH-R的最佳浓度得以维持在质膜上。与细胞系统模型相比，这项在天然环境中使用荧光成像对GHRH/GHRH-R复合物动力学进行的首次研究表明，受体一级结构和质膜浓度在特定G蛋白偶联受体(GPCR)的内化和运输中均起重要作用。
