Authier F, Desbuquois B, De Galle B
INSERM U.30, Hôpital des Enfants-Malades, Paris, France.
Endocrinology. 1992 Jul;131(1):447-57. doi: 10.1210/endo.131.1.1319325.
The ligand-induced internalization of the hepatic glucagon receptor has been studied in rats in vivo using cell fractionation. Injection of glucagon (11 nmol/100 g BW) led to a 2- to 3-fold increase in glucagon-binding activity in Golgi-endosomal (GE) fractions along with a 10-20% decrease in binding activity in plasma membrane (PM) fractions. These changes were time and dose dependent, reaching a maximum by 12-24 min and undergoing reversal in 2 h. Glucagon injection also caused a 20% decrease in glucagon binding to the total particulate fraction, which did not occur when binding was measured in the presence of the detergent octylglucoside. The change in glucagon-binding activity in PM and GE fractions resulted mainly from a change in receptor number; affinity remained unaffected (apparent Kd, 0.5 and 5 nM, respectively). A 5- to 10-fold increase in the glucagon content of GE fractions was observed in glucagon-treated rats. Neither the distribution of PM and Golgi marker enzymes nor that of the asialoglycoprotein receptor was affected by glucagon treatment. Regardless of glucagon treatment, glucagon receptors in GE fractions were less sensitive to GTP than receptors in PM fractions with respect to both inhibition of steady state binding and dissociation of prebound ligand. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, glucagon-receptor complexes formed in PM and GE fractions and subsequently cross-linked showed the same apparent mol wt (57 kilodaltons). In addition, they were identically sensitive to N-glycanase treatment, with two major species of lower mol wt generated. However, only cross-linked complexes associated with PM fractions showed detectable GTP sensitivity. GE fractions displayed a GTP-sensitive adenylate cyclase activity that was about 12 times lower than that of PM fractions. In both fractions, activity was stimulated by the addition of forskolin (8-fold) and, to a lesser extent, glucagon (3-fold). In vivo glucagon treatment led to an increase in activity in GE, but not PM, fractions. These results are consistent with the view that upon acute occupancy, hepatic glucagon receptors are rapidly and specifically internalized along with their ligand. During this process, receptor retained structural integrity and uncouple, albeit partially, from other components of the adenylate cyclase system.
利用细胞分级分离技术在大鼠体内研究了配体诱导的肝胰高血糖素受体的内化过程。注射胰高血糖素（11 nmol/100 g体重）导致高尔基体-内体（GE）组分中胰高血糖素结合活性增加2至3倍，同时质膜（PM）组分中的结合活性降低10 - 20%。这些变化具有时间和剂量依赖性，在12 - 24分钟时达到最大值，并在2小时内逆转。注射胰高血糖素还导致与总颗粒组分结合的胰高血糖素减少20%，而在去污剂辛基葡糖苷存在下测量结合时则未出现这种情况。PM和GE组分中胰高血糖素结合活性的变化主要源于受体数量的改变；亲和力未受影响（表观Kd分别为0.5和5 nM）。在接受胰高血糖素治疗的大鼠中，观察到GE组分中胰高血糖素含量增加了5至10倍。胰高血糖素治疗对PM和高尔基体标记酶的分布以及去唾液酸糖蛋白受体的分布均无影响。无论是否进行胰高血糖素治疗，GE组分中的胰高血糖素受体在稳态结合抑制和预结合配体解离方面对GTP的敏感性均低于PM组分中的受体。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，在PM和GE组分中形成并随后交联的胰高血糖素-受体复合物显示出相同的表观分子量（57千道尔顿）。此外，它们对N-糖苷酶处理的敏感性相同，产生了两种较低分子量的主要物种。然而，只有与PM组分相关的交联复合物显示出可检测的GTP敏感性。GE组分显示出对GTP敏感的腺苷酸环化酶活性，比PM组分低约12倍。在两个组分中，活性均受到福斯可林添加的刺激（8倍），以及在较小程度上受到胰高血糖素的刺激（3倍）。体内胰高血糖素治疗导致GE组分而非PM组分中的活性增加。这些结果与以下观点一致，即急性占据后，肝胰高血糖素受体与其配体一起迅速且特异性地内化。在此过程中，受体保持结构完整性，并与腺苷酸环化酶系统的其他组分部分解偶联。
