Mapping the binding domains of the alpha(IIb) subunit. A study performed on the activated form of the platelet integrin alpha(IIb)beta(3).
alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.
Biris
Nikolaos
N
Department of Chemistry, University of Ioannina, Ioannina, Greece.
Abatzis
Morfis
M
Mitsios
John V
JV
Sakarellos-Daitsiotis
Maria
M
Sakarellos
Constantinos
C
Tsoukatos
Demokritos
D
Tselepis
Alexandros D
AD
Michalis
Lambros
L
Sideris
Dimitrios
D
Konidou
Georgia
G
Soteriadou
Ketty
K
Tsikaris
Vassilios
V
D000244:Adenosine Diphosphate / Q000494:pharmacology; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D054641:Dual Specificity Phosphatase 2; D005340:Fibrinogen / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054648:Protein Phosphatase 2; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D017027:Protein Tyrosine Phosphatases / Q000187:drug effects / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D021122:Protein Subunits; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D054648:Protein Phosphatase 2; C517032:DUSP2 protein, human; D054641:Dual Specificity Phosphatase 2; C517033:Dusp2 protein, mouse; D017027:Protein Tyrosine Phosphatases; D019793:Fluorescein