Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway.
P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.
Disdier
M
M
Department of Medicine, St. Francis Medical Research Institute, University of Oklahoma Health Sciences Center, Oklahoma City.
Morrissey
J H
JH
Fugate
R D
RD
Bainton
D F
DF
McEver
R P
RP
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D051379:Mice; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000235:genetics
D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D021382:Protein Sorting Signals; D000324:Adrenocorticotropic Hormone