The membrane-proximal intermolecular disulfide bonds in glycoprotein Ib influence receptor binding to von Willebrand factor.
BACKGROUND
In the platelet glycoprotein (GP)Ib-IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted to the N-terminal domain of the GPIbalpha subunit. How the other subunits in the complex, GPIbbeta and GPIX, regulate the GPIbalpha-VWF interaction is not clear.


OBJECTIVE
As GPIbalpha connects with two GPIbbeta subunits via disulfide bonds, we tested whether these intersubunit covalent links were important to the proper VWF-binding activity of the GPIb-IX complex by characterizing the structure and VWF-binding activity of a mutant GPIb-IX complex that lacked the GPIbalpha-GPIbbeta disulfide bonds.


RESULTS
Mutating both Cys484 and Cys485 of GPIbalpha to serine prevents GPIbalpha from forming covalent disulfide bonds with GPIbbeta, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIbbeta subunits to form a disulfide bond between themselves. As compared to Chinese hamster ovary (CHO) cells stably expressing the wild-type GPIb-IX complex at a comparable level, CHO cells stably expressing the mutant GPIb-IX complex bind to significantly less soluble VWF in the presence of ristocetin and roll on the immobilized VWF under flow at a higher velocity.


CONCLUSIONS
The disulfide bonds between GPIbalpha and GPIbbeta are necessary for optimal GPIbalpha binding to VWF. The structural plasticity around the disulfide bonds may also help to shed light on the inside-out mechanism underlying GPIbbeta modulation of VWF binding.
Mo
X
X
Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
Luo
S-Z
SZ
Munday
A D
AD
Sun
W
W
Berndt
M C
MC
López
J A
JA
Dong
J-F
JF
Li
R
R
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine; D004220:Disulfides; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D010477:Perfusion; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D014162:Transfection; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D050505:Mutant Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D021122:Protein Subunits; D014841:von Willebrand Factor; D003545:Cysteine