Unique disulfide bonds in epidermal growth factor (EGF) domains of β3 affect structure and function of αIIbβ3 and αvβ3 integrins in different manner.
The β3 subunit of αIIbβ3 and αvβ3 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 αIIbβ3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbβ3 and αvβ3. We created β3 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 αv or αIIb. Flow cytometry was used to measure surface expression of αIIbβ3 and αvβ3 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 αIIbβ3 and αvβ3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbβ3 and αvβ3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbβ3. Activation of C523S/C544S αvβ3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S β3 in αIIbβ3 but not in αvβ3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in β3 differently affect the function of αIIbβ3 and αvβ3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbβ3 and αvβ3 and for Cys-523-Cys-544 only in αvβ3.
the Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer, Israel. ronit.cohen@sheba.health.gov.il
Ronit
R
Mor-Cohen
Nurit
N
Rosenberg
Yulia
Y
Einav
Ehud
E
Zelzion
Meytal
M
Landau
Wissam
W
Mansour
Yulia
Y
Averbukh
Uri
U
Seligsohn
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D004220:Disulfides; D039302:Integrin alphaVbeta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D004815:Epidermal Growth Factor