Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta(3) integrins following disulfide bond disruption by cysteine mutations - Identification of cysteine 598 involved in alpha(IIb)beta(3) activation

We have investigated receptor function and epitope expression of recombinan t alpha (IIb)beta (3) mutated at Cys(177) or Cys(273) in the I-like domain as well as Cys(598), located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II pa tient. The beta (3) mutants beta (3)C177A, beta (3)C273A, and beta (3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel e lectrophoresis under nonreducing conditions, confirming the disruption of t he respective disulfide loops. Despite reduced surface expression, the alph a (IIb)beta (3)C177A, alpha (IIb)beta (3)C273A, and alpha (IIb)beta (3)C598 Y receptors mediated cell adhesion to immobilized fibrinogen and translocat ed into focal adhesion plaques. The beta (3)C598Y mutation, but not the bet a (3)C177A or beta (3)C273A mutations, induced spontaneous binding of the l igand mimetic monoclonal antibody PAC-1, while the beta (3)C177A and beta ( 3)C273A mutants exhibited reduced complex stability in the absence of Ca2+. Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, p12-46, 10E5, and P256 did not interact with alpha (IIb)beta (3)C273A and bound only weakly t o alpha (IIb)beta (3)C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant alpha (IIb)beta (3)C273A, but interacted only weakly with mutant al pha (IIb)beta (3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D 10G3 and not exposed on wild type alpha (IIb)beta (3) became accessible onl y on mutant alpha (IIb)beta (3)C177A and was mapped to the 60-kDa chymotryp sin fragment of beta (3). Finally, the ligand-induced binding site (LIBS) e pitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provi de evidence that disruption of a single cysteine disulfide bond in the cyst eine-rich repeat domain, but not in the I-like domain, activates integrin a lpha (IIb)beta (3). In contrast, disruption of each of the disulfide bonds in the two long insertions of the I-like domain predicted to be in close co ntact with the alpha subunit beta -propeller domain affect the stability of the alpha (IIb)beta (3) heterodimer and inhibit complex-specific mAb bindi ng without affecting the RGD binding capacity of the metal ion-dependent ad hesion site-like domain.