A single amino acid substitution flanking the fourth calcium binding domain of alpha IIb prevents maturation of the alpha IIb beta 3 integrin complex.
To define specific structural domains involved in the biosynthesis and processing of integrin subunits, we examined the biosynthesis of normal and mutated forms of the platelet-specific integrin alpha IIb beta 3. Platelet mRNA was isolated from a Glanzmann thrombasthenic patient who failed to express significant levels of the glycoprotein (GP) IIb-IIIa complex on the platelet surface. Sequence analysis of polymerase chain reaction-amplified platelet GPIIb mRNA revealed a Gly418-->Asp amino acid substitution in GPIIb. Gly418 is a highly conserved residue that flanks the fourth calcium binding domain of GPIIb. Cotransfection of Asp418 GPIIb and GPIIIa plasmid constructs into COS-7 cells resulted in the accumulation of a pre-GPIIb-IIIa complex that failed to reach the cell surface, in effect recreating the thrombasthenic phenotype. Pulse-chase and endoglycosidase studies demonstrated that the biosynthetic blockade occurred in a pre-Golgi compartment. Removal of the negatively charged carboxyl group of Asp418 GPIIb, creating Ala418 GPIIb, rescued intracellular transport and surface expression of the integrin complex. Mutagenesis of a homologous Gly within the integrin alpha subunit alpha v also resulted in the failure to express alpha v beta 3 on the cell surface. These results suggest that the presence of a small, uncharged amino acid 6-8 residues amino-terminal to the calcium coordination complex is crucial for the proper folding and maturation of integrin complexes.
Wilcox
D A
DA
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee 53233.
Wautier
J L
JL
Pidard
D
D
Newman
P J
PJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D005998:Glycine / Q000737:chemistry; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013329:Structure-Activity Relationship; D013915:Thrombasthenia / Q000235:genetics; D014162:Transfection
D017931:DNA Primers; D016023:Integrins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D002118:Calcium; D005998:Glycine