P-Selectin, A Granule Membrane Protein of Platelets and Endothelial Cells, Follows the Regulated Secretory Pathway in AtT-20 Cells

• Published in: The Journal of cell biology Vol. 116; no. 3; pp. 617 - 625
• Main Authors: Koedam, Joost A., Cramer, Elisabeth M., Briend, Emmanuel, Furie, Bruce, Furie, Barbara C., Wagner, Denisa D.
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.02.1992; The Rockefeller University Press
• Subjects: 8-Bromo Cyclic Adenosine Monophosphate - pharmacology; Adrenocorticotropic Hormone - analysis; Adrenocorticotropic Hormone - metabolism; Animals; Antibodies; Biological and medical sciences; Cell Adhesion Molecules - analysis; Cell Adhesion Molecules - metabolism; Cell Fractionation; Cell Line; Cell lines; Cell Membrane - metabolism; Cell membranes; Cell physiology; Cells; CHO cells; Cytoplasmic Granules - chemistry; Cytoplasmic Granules - metabolism; Cytoplasmic Granules - ultrastructure; Endothelial cells; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Membrane proteins; Microscopy, Immunoelectron; Molecular and cellular biology; P-Selectin; pituitary; Platelet Membrane Glycoproteins - analysis; Platelet Membrane Glycoproteins - metabolism; Platelets; Secretion; Secretion. Exocytosis; Secretory vesicles; Transfection; Transmembrane protein; Endothelial cell; Secretion; Rodentia; Molecular targeting; Vertebrata; Platelet; Mammalia; Cell line; Mouse; Immunoelectron microscopy; Immunofluorescence; Localization
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.116.3.617

P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to α granules of platelets and Weibel-Palade bodies of endothelial 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.