P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells.
P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to alpha granules of platelets and Weibel-Palade bodies of endotheial cells. Upon stimulation of these cells, P-selectin is translocated to the plasma membrane where it functions as a receptor for monocytes and neutrophils. To investigate whether the mechanism of targeting of P-selectin to granules is specific for megakaryocytes and endothelial cells and/or dependent on von Willebrand factor, a soluble adhesive protein that is stored in the same granules, we have expressed the cDNA for P-selectin in AtT-20 cells. AtT-20 cells are a mouse pituitary cell line that can store proteins in a regulated fashion. By double-label immunofluorescence, P-selectin was visible as a punctate pattern at the tips of cell processes. This pattern closely resembled the localization of ACTH, the endogenous hormone produced and stored by the AtT-20 cells. Fractionation of the transfected cells resulted in the codistribution of P-selectin and ACTH in cellular compartments of the same density. Immunoelectron microscopy using a polyclonal anti-P-selectin antibody demonstrated immunogold localization in dense granules, morphologically indistinguishable from the ACTH granules. Binding experiments with radiolabeled monoclonal antibody to P-selectin indicated that there was also surface expression of P-selectin on the AtT-20 cells. After stimulation with the secretagogue 8-Bromo-cAMP the surface expression increased twofold, concomitant with the release of ACTH. In contrast, the surface expression of P-selectin transfected into CHO cells, which do not have a regulated pathway of secretion, did not change with 8-Br-cAMP treatment. In conclusion, we provide evidence for the regulated secretion of a transmembrane protein (P-selectin) in a heterologous cell line, which indicates that P-selectin contains an independent sorting signal directing it to storage granules.
Department of Medicine, Boston, Massachusetts.
J A
JA
Koedam
E M
EM
Cramer
E
E
Briend
B
B
Furie
B C
BC
Furie
D D
DD
Wagner
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000324:Adrenocorticotropic Hormone / Q000032:analysis / Q000378:metabolism; D000818:Animals; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D016253:Microscopy, Immunoelectron; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism*; D014162:Transfection
D015815:Cell Adhesion Molecules; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000324:Adrenocorticotropic Hormone