Merlen Clémence, Fabrega Sylvie, Desbuquois Bernard, Unson Cecilia G, Authier François
Institut National de la Santé et de la Recherche Médicale Unité 756, Faculté de Pharmacie Paris XI, 92296 Châtenay-Malabry, France.
FEBS Lett. 2006 Oct 16;580(24):5697-704. doi: 10.1016/j.febslet.2006.09.021. Epub 2006 Sep 20.
To assess glucagon receptor compartmentalization and signal transduction in liver parenchyma, we have studied the functional relationship between glucagon receptor endocytosis, phosphorylation and coupling to the adenylate cyclase system. Following administration of a saturating dose of glucagon to rats, a rapid internalization of glucagon receptor was observed coincident with its serine phosphorylation both at the plasma membrane and within endosomes. Co-incident with glucagon receptor endocytosis, a massive internalization of both the 45- and 47-kDa Gsalpha proteins was also observed. In contrast, no change in the subcellular distribution of adenylate cyclase or beta-arrestin 1 and 2 was observed. In response to des-His(1)-[Glu(9)]glucagon amide, a glucagon receptor antagonist, the extent and rate of glucagon receptor endocytosis and Gsalpha shift were markedly reduced compared with wild-type glucagon. However, while the glucagon analog exhibited a wild-type affinity for endosomal acidic glucagonase activity and was processed at low pH with similar kinetics and rates, its proteolysis at neutral pH was 3-fold lower. In response to tetraiodoglucagon, a glucagon receptor agonist of enhanced biological potency, glucagon receptor endocytosis and Gsalpha shift were of higher magnitude and of longer duration, and a marked and prolonged activation of adenylate cyclase both at the plasma membrane and in endosomes was observed. The subsequent post-endosomal fate of internalized Gsalpha was evaluated in a cell-free rat liver endosome-lysosome fusion system following glucagon injection. A sustained endo-lysosomal transfer of the two 45- and 47-kDa Gsalpha isoforms was observed. Therefore, these results reveal that within hepatic target cells and consequent to glucagon-mediated internalization of the serine-phosphorylated glucagon receptor and the Gsalpha protein, extended signal transduction may occur in vivo at the locus of the endo-lysosomal apparatus.
为了评估肝脏实质中胰高血糖素受体的区室化和信号转导，我们研究了胰高血糖素受体的内吞作用、磷酸化以及与腺苷酸环化酶系统偶联之间的功能关系。给大鼠注射饱和剂量的胰高血糖素后，观察到胰高血糖素受体迅速内化，同时在质膜和内体中其丝氨酸发生磷酸化。与胰高血糖素受体内吞作用同时发生的是，还观察到45 kDa和47 kDa的Gsα蛋白大量内化。相比之下，未观察到腺苷酸环化酶或β-抑制蛋白1和2的亚细胞分布发生变化。与胰高血糖素受体拮抗剂去组氨酸-[谷氨酸(9)]胰高血糖素酰胺反应时，与野生型胰高血糖素相比，胰高血糖素受体的内吞程度和速率以及Gsα的移位明显降低。然而，虽然该胰高血糖素类似物对内体酸性胰高血糖素酶活性具有野生型亲和力，并且在低pH下以相似的动力学和速率进行加工，但其在中性pH下的蛋白水解作用降低了3倍。与生物活性增强的胰高血糖素受体激动剂四碘胰高血糖素反应时，胰高血糖素受体的内吞作用和Gsα移位幅度更大、持续时间更长，并且在质膜和内体中均观察到腺苷酸环化酶的显著且持久的激活。在注射胰高血糖素后，在无细胞的大鼠肝脏内体-溶酶体融合系统中评估内化的Gsα的内体后命运。观察到两种45 kDa和47 kDa的Gsα同工型持续进行内体-溶酶体转移。因此，这些结果表明，在肝靶细胞内，由于胰高血糖素介导的丝氨酸磷酸化的胰高血糖素受体和Gsα蛋白的内化，体内可能在内体-溶酶体装置部位发生延长的信号转导。
