Identification of the region in actin-binding protein that binds to the cytoplasmic domain of glycoprotein IBalpha.
Actin-binding protein (ABP-280) is a component of the submembranous cytoskeleton and interacts with the glycoprotein (GP) Ibalpha subunit of the GP Ib-IX complex in platelets. In the present studies, we have identified the binding site for GP Ibalpha in ABP-280. A melanoma cell line lacking ABP-280 was stably transfected with the cDNAs coding for GP Ib-IX, then transiently transfected with cDNA coding for various carboxyl-truncates of ABP-280. Immunocapture assays and co-immunoprecipitation experiments from detergent-lysed cells showed that deletion of the carboxyl-terminal repeats 20-24 of ABP-280 had no effect on GP Ib-IX binding, but deletion of residues 2099 through 2136 within repeat 19 abolished binding. In the yeast two-hybrid system, an ABP-280 fragment comprising repeats 17-19 bound GP Ibalpha. Deletion from either end abolished binding. Individual or multiple repeats of ABP-280 were expressed as fusion protein in bacteria and purified; structural folding was evaluated, and binding to GP Ib-IX was assessed. Binding depended on the presence of repeats 17-19. None of the individual repeats were able to bind to GP Ib-IX. These findings demonstrate that residues 1850-2136 comprising repeats 17-19 contain the binding site for GP Ib-IX.
Meyer
S C
SC
Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Zuerbig
S
S
Cunningham
C C
CC
Hartwig
J H
JH
Bissell
T
T
Gardner
K
K
Fox
J E
JE
D001665:Binding Sites; D002460:Cell Line; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006801:Humans; D008545:Melanoma; D008840:Microfilament Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000096:biosynthesis
D008840:Microfilament Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase