A deletion in the alpha subunit locks platelet integrin alpha IIb beta 3 into a high affinity state.
The integrin alpha IIb beta 3 (GPIIb/IIIa) mediates platelet aggregation by a change in affinity for the ligand fibrinogen. The amino acids 991-995 (GFFKR) at the NH2-terminus of the cytoplasmic domain are highly conserved in all known integrin alpha subunits. We postulated that the GFFKR-region is important for the inside-out signal transduction and has an influence on the affinity state of integrins. To test this hypothesis, a mutant with a deletion in the GFFKR region was designed. The DNA-constructs were constructed by PCR, sequenced, cotransfected with the beta 3 subunit into CHO cells and cell surface expression was proven with immunoprecipitation and flow cytometry. The GFFKR-deletion mutant demonstrated a high affinity binding of the mAb PAC-1 and I125-labeled fibrinogen. The metabolic inhibitors 2-deoxyglucose and NaN3 did not change the affinity state of the deleted receptor. Neither did the truncation of the cytoplasmic domain of the beta 3 subunit. Additionally, expression of the deleted integrin in the erythropoetic cell line K562 revealed a high affinity state. A deletion of the GFFKR-region in the cytoplasmic domain of the alpha subunit locks integrin alpha IIb beta 3 in a high affinity state. This is an intrinsic property of the deleted receptor since there is no energy dependence and no cell type specifity. Thus, the GFFKR-region is involved in inside-out signaling in alpha IIb beta 3. Furthermore, cell lines expressing this activated alpha IIb beta 3 integrin may be used as models for activated platelets.
Peter
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Department of Cardiology, University of Heidelberg, Germany. kpeter@krzmail.krz.uni-heidelberg.de
Bode
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D011487:Protein Conformation; D014162:Transfection
D018076:DNA, Complementary; D019039:Platelet Glycoprotein GPIIb-IIIa Complex