Specific cysteines in β3 are involved in disulfide bond exchange-dependent and -independent activation of αIIbβ3

Ronit Mor-Cohen, Nurit Rosenberg, Meytal Landau, Judith Lahav, Uri Seligsohn

Research output: Contribution to journalArticlepeer-review

Abstract

Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of β3 was suggested to be involved in activation of αIIbβ3. To investigate the role of specific β3 cysteines in αIIbβ3 expression and activation, we expressed in baby hamster kidney cells normal αIIb with normal β3 or β3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and β-tail domains and assessed αIIbβ3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of αIIbβ3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active αIIbβ3, implying that these bonds stabilize the inactive αIIbβ3 conformer. Mutants of the Cys-567-Cys-581 bond in EGF-4 were inactive even after exposure to αIIbβ3-activating antibodies, indicating that this bond is necessary for activating αIIbβ3. Disrupting Cys-560-Cys-583 in the EGF-3/EGF-4 or Cys-608-Cys-655 in β-tail domain resulted in αIIbβ3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in αIIbβ3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type αIIbβ3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in αIIbβ3 activation. These data suggest that different disulfide bonds in β3 EGF and β-tail domains play variable structural and regulatory roles in αIIbβ3.

Original languageEnglish
Pages (from-to)19235-19244
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number28
DOIs
StatePublished - 11 Jul 2008

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