Mutation of a Ligand Binding Domain of β₃ Integrin: Integral Role of Oxygenated Residues in α_IIbβ₃ (GPIIb-IIIa) Receptor Function

A single amino acid substitution in β₃ (Asp¹¹⁹ → Tyr) abrogates the ligand binding function of β₃ integrins and alters the divalent cation conformation of the platelet integrin α_IIbβ₃ (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 β₃ 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 β₃ 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 α_IIbβ₃-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 α_IIbβ₃.