Effect of deletion of glycoprotein IIb exon 28 on the expression of the platelet glycoprotein IIb/IIIa complex.
We have isolated from an HEL cell cDNA library an alternatively spliced transcript for the platelet membrane glycoprotein IIb (GPIIb) that resulted from the deletion of the 34 amino acids of exon 28 of the GPIIb gene. Confirming an earlier report, we also detected this transcript in platelet mRNA. To determine the consequences of exon 28 deletion on the expression of the GPIIb/IIIa heterodimer, we expressed cDNA for GPIIb-28 in COS-1 cells, either individually or simultaneously with a cDNA for GPIIIa. When recombinant GPIIb-28 was expressed alone, it did not acquire resistance to the enzyme endo-beta-N-acetylglucosaminidase H, was not cleaved into heavy and light chains, and was not transported to the cell surface. However, when recombinant GPIIb-28 was coexpressed with recombinant GPIIIa, GPIIb/IIIa heterodimers were assembled. Nevertheless, these heterodimers failed to complete posttranslational processing and were degraded intracellularly. Exon 28 contains one site for Asn-linked glycosylation. To determine if loss of this glycosylation site was responsible for the effects of exon 28 deletion, we removed the site from the exon 28 of intact GPIIb by oligonucleotide-mediated mutagenesis. However, absence of the carbohydrate appended to exon 28 did not prevent normal GPIIb/IIIa heterodimer expression. Our studies indicate that absence of the amino acids encoded by GPIIb exon 28 sufficiently perturbs the quaternary configuration of the GPIIb/IIIa heterodimer to impair its subsequent intracellular transport and processing. They also indicate that this alternatively spliced form of GPIIb mRNA, although present in megakaryocytes, is unlikely to make a significant contribution to the GPIIb/IIIa complexes expressed on platelets.
Hematology-Oncology Section, Hospital of the University of Pennsylvania, Philadelphia 19104.
M A
MA
Kolodziej
G
G
Vilaire
S
S
Rifat
M
M
Poncz
J S
JS
Bennett
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002872:Chromosome Deletion; D005091:Exons / Q000235:genetics*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010980:Platelet Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins