Determinants of the intracellular fate of truncated forms of the platelet glycoproteins IIb and IIIa.
The platelet glycoproteins GPIIb and GPIIIa are integral membrane proteins and form calcium-dependent heterodimers in the endoplasmic reticulum (ER). In the absence of heterodimer formation, GPIIb and GPIIIa are retained in the ER and degraded. To produce soluble forms of these proteins, we truncated each at a site just proximal to its transmembrane anchor and expressed the mutants in COS-1 cells. We found that both truncated GPIIIa (GPIIIatr) and GPIIIatr were secreted by the transfected cells. However, GPIIbtr was retained by the cells and was immunoprecipitated as a doublet with a 115,000 molecular weight protein. Incubation of transfected cells with the calcium ionophore A23187 or the calcium chelator 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) failed to induce appreciable GPIIbtr secretion, suggesting that formation of intracellular calcium complexes was not a factor in GPIIbtr retention. Further, immunoblotting of immunoprecipitated GPIIbtr and GPIIIatr revealed that the chaperone binding protein (BiP) was associated with each, arguing that BiP alone was not responsible for GPIIbtr retention. These studies indicate that the intracellular retention of GPIIIa involves sequences located in the transmembrane or cytoplasmic domains of the molecule. GPIIb contains an additional retention signal located in the extracellular portion of the molecule whose effect is abrogated by formation of a GPIIb-IIIa heterodimer. This signal may be involved in the fate of nascent GPIIb monomers and the generation of correctly configured GPIIb-IIIa heterodimers.
Bennett
J S
JS
Hematology-Oncology Division, Hospital of the University of Pennsylvania, Philadelphia 19104.
Kolodziej
M A
MA
Vilaire
G
G
Poncz
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D000001:Calcimycin; D004533:Egtazic Acid; D004247:DNA; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid