A chimeric proinsulin-CD5 protein expressed in AtT-20 cells is directed to the cell surface via the constitutive pathway.
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.
Mbikay
M
M
Laboratoire de Neuroendocrinologie Moléculaire, Institut de Recherches Cliniques de Montréal, Université de Montréal, Québec, Canada.
Grondin
G
G
Rondeau
N
N
Talbot
B G
BG
Chrétien
M
M
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D018956:CD5 Antigens; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D012313:RNA; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D015703:Antigens, CD; D018956:CD5 Antigens; D007328:Insulin; D008565:Membrane Proteins; C039919:RNA, recombinant; D011993:Recombinant Fusion Proteins; D012313:RNA; D011384:Proinsulin