Unique disulfide bonds in epidermal growth factor (EGF) domains of ?3 affect structure and function of ?IIb?3 and ?v?3 integrins in different manner.

ABSTRACT: The ?3 subunit of ?IIb?3 and ?v?3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for ?IIb?3 activation. In this study we further investigated the role of all four integrin unique bonds in both ?IIb?3 and ?v?3. We created ?3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal ?v or ?IIb. Flow cytometry was used to measure surface expression of ?IIb?3 and ?v?3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of ?IIb?3 and ?v?3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active ?IIb?3 and ?v?3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active ?IIb?3. Activation of C523S/C544S ?v?3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S ?3 in ?IIb?3 but not in ?v?3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in ?3 differently affect the function of ?IIb?3 and ?v?3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both ?IIb?3 and ?v?3 and for Cys-523-Cys-544 only in ?v?3.

SUBMITTER: Mor-Cohen R 

PROVIDER: S-EPMC3308749 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature