Processing and assembly of the integrin, glycoprotein IIb-IIIa, in HEL cells.
We examined the biosynthetic processing and assembly of the platelet glycoprotein (GP) IIb-IIIa complex in [35S]methionine-labeled HEL cells, a human cell line with features of megakaryocytes. Both GPIIb and GPIIIa were synthesized as single-chain precursors to which high mannose N-linked oligosaccharides were added in the endoplasmic reticulum (ER). A 5-fold excess of the major IIb precursor, preIIb, was synthesized relative to GPIIIa. Two smaller proteins immunologically related to GPIIb were synthesized in smaller amounts. Assembly of the GPIIb and GPIIIa precursors required 4-6 h for completion. All GPIIIa molecules were eventually assembled; the excess GPIIb precursors were degraded without reaching the cell surface. Following assembly, preIIb-IIIa complexes were rapidly transported to the Golgi apparatus where preIIb underwent modification of high mannose chains into complex oligosaccharides and proteolytic cleavage to yield disulfide-linked heavy and light chains. Pretreating cells with the ionophore monensin blocked cleavage of preIIb but not its carbohydrate modification or its assembly with GPIIIa. These studies suggest that 1) assembly of the precursors of GPIIb and GPIIIa in the ER is a slow process requiring conformational maturation of one or both subunits, and 2) only heterodimers assembled in the ER are transported to the Golgi apparatus for additional processing and, ultimately, expression on the cell surface.
Rosa
J P
JP
Institut National de la Santé et de la Recherche Médicale, Paris, France.
McEver
R P
RP
D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D016023:Integrins; D007700:Kinetics; D008533:Megakaryocytes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008715:Methionine / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013462:Sulfur Radioisotopes
D000911:Antibodies, Monoclonal; D016023:Integrins; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D013462:Sulfur Radioisotopes; D008985:Monensin; D004492:Edetic Acid; D008715:Methionine