Tyr178 of beta3 is critical for alphaIIb maturation and macromolecular ligand binding to alphaIIbbeta3.
To explore the structural basis of ligand binding to alphaIIbbeta3, we conducted a site-directed mutagenesis of Y178, which is located in the ligand-specificity region (C177-C184) of the beta3 subunit. Two mutant beta3 constructs, Y178A and Y178I, were transfected into CHO cells and co-expressed with human alphaIIb subunit on the cell surface. Our results showed that the Y178A mutation affected processing and cell surface exposure of recombinant alphaIIbbeta3 receptor, abrogated the binding of PAC-1, a ligand-mimetic antibody, to alphaIIbbeta3 pre-treated with the known activator DTT. The Y178A mutation also resulted in reduced adhesion of alphaIIbbeta3 on immobilized fibrinogen. In contrast, the interaction of alphaIIbbeta3 with the small molecular ligand RGDS was unaffected by Y178A mutation, as evidenced by the elevated LIBS-1 epitope expression following RGDS addition. Interestingly however, Y178I mutation did not affect the receptor synthesis and function at all. As for post-receptor occupancy, neither Y178A nor Y178I prevented alphaIIbbeta3 translocation to focal adhesion contacts. These results suggest that Y178 is involved in alphaIIb maturation and alphaIIbbeta3 complex expression. This residue is also critical for alphaIIbbeta3 interaction with its macromolecular ligand or ligand-mimetic mAb, possibly due to its involvement in other ligand-binding sites distinct from the RGD-binding pocket. We also propose that a residue with appropriate side-chain size and hydrophobicity at position 178 is spatially required for formation of the correct tertiary structure of the site.
Sun
Chong-Xiu
CX
Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, Nanjing, 210093, China.
Chen
Ping
P
Lu
Wei
W
Liu
Jian-Ning
JN
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry; D006801:Humans; D008024:Ligands; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
D000939:Epitopes; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D014443:Tyrosine; D005340:Fibrinogen