Regazzi R, Ravazzola M, Iezzi M, Lang J, Zahraoui A, Andereggen E, Morel P, Takai Y, Wollheim C B
Department of Medicine, University of Geneva, Switzerland (Member of the Geneva Diabetes Group).
J Cell Sci. 1996 Sep;109 ( Pt 9):2265-73. doi: 10.1242/jcs.109.9.2265.
We examined the presence of small molecular mass GTP-binding proteins of the Rab3 family in different insulin-secreting cells. Rab3B and Rab3C were identified by western blotting in rat and in human pancreatic islets, in two rat insulin-secreting cell lines, RINm5F and INS-1, as well as in the hamster cell line HIT-T15. In contrast, Rab3A was detected in rat pancreatic islets as well as in the two insulin-secreting rat cell lines but not in human pancreatic islets and was only barely discernible in HIT-T15 cells. These findings were confirmed by two-dimensional gel electrophoresis followed by GTP-overlay of homogenates of pancreatic islets and of the purified protein. Northern blotting analysis revealed that Rab3D is expressed in the same insulin-secreting cells as Rab3A. Separation of the cells of the rat islets by fluorescence-activated cell sorting demonstrated that Rab3A was exclusively expressed in beta-cells. Rab3A was found to be associated with insulin-containing secretory granules both by immunofluorescence, immunoelectron microscopy and after sucrose density gradient. Overexpression in HIT-T15 cells of a Rab3A mutant deficient in GTP hydrolysis inhibited insulin secretion stimulated by a mixture of nutrients and bombesin. Insulin release triggered by these secretagogues was also slightly decreased by the overexpression of wild-type Rab3A but not by the overexpression of wild-type Rab5A and of a Rab5A mutant deficient in GTP hydrolysis. Finally, we studied the expression in insulin-secreting cells of rabphilin-3A, a putative effector protein that associates with the GTP-bound form of Rab3A. This Rab3A effector was not detectable in any of the cells investigated in the present study. Taken together these results indicate an involvement of Rab3A in the control of insulin release in rat and hamster. In human beta-cells, a different Rab3 isoform but with homologous function may replace Rab3A.
我们检测了Rab3家族小分子质量GTP结合蛋白在不同胰岛素分泌细胞中的存在情况。通过蛋白质印迹法在大鼠和人胰岛、两种大鼠胰岛素分泌细胞系RINm5F和INS-1以及仓鼠细胞系HIT-T15中鉴定出了Rab3B和Rab3C。相比之下，在大鼠胰岛以及两种大鼠胰岛素分泌细胞系中检测到了Rab3A，但在人胰岛中未检测到，且在HIT-T15细胞中仅勉强可辨。胰岛匀浆和纯化蛋白的二维凝胶电泳随后进行GTP覆盖证实了这些发现。Northern印迹分析显示，Rab3D与Rab3A在相同的胰岛素分泌细胞中表达。通过荧光激活细胞分选分离大鼠胰岛细胞表明，Rab3A仅在β细胞中表达。通过免疫荧光、免疫电子显微镜以及蔗糖密度梯度离心发现Rab3A与含胰岛素的分泌颗粒相关。在HIT-T15细胞中过表达缺乏GTP水解能力的Rab3A突变体抑制了由营养物质和蛙皮素混合物刺激的胰岛素分泌。这些促分泌剂引发的胰岛素释放也因野生型Rab3A的过表达而略有降低，但野生型Rab5A以及缺乏GTP水解能力的Rab5A突变体的过表达则没有这种作用。最后，我们研究了rabphilin-3A在胰岛素分泌细胞中的表达，rabphilin-3A是一种假定的效应蛋白，与Rab3A的GTP结合形式相关。在本研究中所调查的任何细胞中均未检测到这种Rab3A效应蛋白。综合这些结果表明，Rab3A参与大鼠和仓鼠胰岛素释放的调控。在人β细胞中，可能有不同但功能同源的Rab3同工型替代Rab3A。
