Mutation of a ligand binding domain of beta 3 integrin. Integral role of oxygenated residues in alpha IIb beta 3 (GPIIb-IIIa) receptor function.
A single amino acid substitution in beta 3 (Asp119 --> Tyr) abrogates the ligand binding function of beta 3 integrins and alters the divalent cation conformation of the platelet integrin alpha IIb beta 3 (GPIIb-IIIa). This aspartic acid residue resides within a conserved cluster of oxygenated residues that may provide ligands for the coordination of divalent cations. To assign function to the other oxygenated residues in this group (Ser121, Ser123, Asp126, Asp127, and Ser130), each of these amino acids in beta 3 was individually substituted by alanine. None of these amino acid substitutions altered heterodimer formation or surface expression. However, the substitutions had differential effects on receptor function. Substitution at positions Asp119 or Ser121 produced a complete loss of receptor function. Cells expressing these mutants failed to adhere to fibrinogen, failed to bind activation-independent ligand-mimetic peptides, and did not bind the ligand-mimetic mAb PAC1 following activation of the receptor. Similarly, cells expressing beta 3 with a substitution at Ser123 also failed to adhere to fibrinogen and did not bind RGD peptide or mAb PAC1. These cells did retain the capacity to bind an alpha IIb beta 3-specific, high affinity peptidomimetic, but occupancy did not induce the conformational change from resting to activated state observed following occupancy of the wild type receptor. Substitution at positions Asp126, Asp127, or Ser130 had no effect on ligand binding function. These data indicate that Asp119, along with Ser121 and Ser123, plays an integral role in the ligand binding function of alpha IIb beta 3.
Bajt
M L
ML
Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037.
Loftus
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites / Q000235:genetics; D001666:Binding Sites, Antibody; D016466:CHO Cells; D017124:Conserved Sequence; D006224:Cricetinae; D005340:Fibrinogen / Q000378:metabolism; D039661:Integrin beta3; D016023:Integrins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D039661:Integrin beta3; D016023:Integrins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen; D010100:Oxygen