Mbikay M, Grondin G, Rondeau N, Talbot B G, Chrétien M
Laboratoire de Neuroendocrinologie Moléculaire, Institut de Recherches Cliniques de Montréal, Université de Montréal, Québec, Canada.
Exp Cell Res. 1995 Sep;220(1):79-91. doi: 10.1006/excr.1995.1294.
A chimeric gene encoding mouse proinsulin fused to the transmembrane and the cytoplasmic domains of the CD5 antigen of human T lymphocytes was expressed in AtT-20 cells to assess the relative strength of signals that influence the sorting of secretory proteins to the regulated or constitutive pathway in endocrine cells. Transfected cells expressing the antigen at the surface were purified by fluorescence-activated cell sorting and analyzed by Northern and Western blots. They contained a mRNA of 1.4 kb hybridizing with an insulin cDNA probe and two immunoreactive insulin forms of 21 and 24 kDa, recognizable by antibodies against both insulin and C peptide. The surface density of these antigens was not increased following KCl stimulation of the cells, suggesting that they were not stored within the cells in significant amounts. This was confirmed by immunoelectron microscopy which showed the antigen attached to membranes, in the Golgi, in endosomes, and at the cell surface, but not in secretory granules. These results indicate that the proinsulin-CD5 fusion protein was transported to the cell surface via the constitutive pathway and partly recycled by endocytosis. They also suggest that the signals that direct proinsulin into storage granules may no longer be dominant when fused to transmembrane and cytosolic sequences derived from a constitutively secreted molecule.
编码与人类T淋巴细胞CD5抗原的跨膜和胞质结构域融合的小鼠胰岛素原的嵌合基因在AtT-20细胞中表达，以评估影响内分泌细胞中分泌蛋白分选至调节性或组成性途径的信号的相对强度。通过荧光激活细胞分选纯化在表面表达该抗原的转染细胞，并通过Northern和Western印迹分析。它们含有与胰岛素cDNA探针杂交的1.4 kb mRNA以及两种免疫反应性胰岛素形式，分子量分别为21 kDa和24 kDa，可被抗胰岛素和C肽的抗体识别。细胞经KCl刺激后，这些抗原的表面密度并未增加，表明它们在细胞内的储存量并不显著。免疫电子显微镜证实了这一点，该显微镜显示抗原附着于膜、高尔基体、内体和细胞表面，但不存在于分泌颗粒中。这些结果表明胰岛素原-CD5融合蛋白通过组成性途径转运至细胞表面，并通过内吞作用部分循环利用。它们还表明，当与来自组成性分泌分子的跨膜和胞质序列融合时，将胰岛素原导向储存颗粒的信号可能不再占主导地位。
