title: 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 alphaIIbbeta3 activation.
abstract: We have investigated receptor function and epitope expression of recombinant 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 patient. The beta(3) mutants beta(3)C177A, beta(3)C273A, and beta(3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the alpha(IIb)beta(3)C177A, alpha(IIb)beta(3)C273A, and alpha(IIb)beta(3)C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The beta(3)C598Y mutation, but not the beta(3)C177A or beta(3)C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the beta(3)C177A and beta(3)C273A mutants exhibited reduced complex stability in the absence of Ca(2+). Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2-46, 10E5, and P256 did not interact with alpha(IIb)beta(3)C273A and bound only weakly to 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 alpha(IIb)beta(3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type alpha(IIb)beta(3) became accessible only on mutant alpha(IIb)beta(3)C177A and was mapped to the 60-kDa chymotrypsin fragment of beta(3). Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin alpha(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 contact with the alpha subunit beta-propeller domain affect the stability of the alpha(IIb)beta(3) heterodimer and inhibit complex-specific mAb binding without affecting the RGD binding capacity of the metal ion-dependent adhesion site-like domain.
authors.affiliation: Laboratoire Franco-Luxembourgeois de Recherche Biomédicale (CNRS/CRP-Santé), Centre Universitaire, L-1511 Luxembourg.
authors.forename: P
authors.initials: P
authors.lastname: Chen
authors.forename: C
authors.initials: C
authors.lastname: Melchior
authors.forename: N H
authors.initials: NH
authors.lastname: Brons
authors.forename: N
authors.initials: N
authors.lastname: Schlegel
authors.forename: J
authors.initials: J
authors.lastname: Caen
authors.forename: N
authors.initials: N
authors.lastname: Kieffer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics*; D015153:Blotting, Western; D016466:CHO Cells; D002448:Cell Adhesion; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides; D004789:Enzyme Activation; D000939:Epitopes / Q000737:chemistry; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D039661:Integrin beta3; D008024:Ligands; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics*; D011233:Precipitin Tests; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018076:DNA, Complementary; D004220:Disulfides; D000939:Epitopes; D039661:Integrin beta3; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D005340:Fibrinogen; D003545:Cysteine