Identification of the Region in Actin-binding Protein that Binds to the Cytoplasmic Domain of Glycoprotein Ibα

doi:10.1074/jbc.272.5.2914

Identification of the Region in Actin-binding Protein that Binds to the Cytoplasmic Domain of Glycoprotein Ib_α*

From the^‡ Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195,
^§ Children's Hospital Oakland Research Institute, Oakland, California 94609,
^¶ Cardiovascular Research Institute, Department of Pathology, University of California, San Francisco, San Francisco, California 94143, and
^∥ Experimental Medicine Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

** To whom correspondence should be addressed:
Joseph J. Jacobs Center for Thrombosis and Vascular Biology (FF20), Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195
. Tel.: 216-445-3874; Fax: 216-445-2051.

Abstract

Actin-binding protein (ABP-280) is a component of the submembranous cytoskeleton and interacts with the glycoprotein (GP) Ib_α subunit of the GP Ib-IX complex in platelets. In the present studies, we have identified the binding site for GP Ib_α 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 Ib_α. 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.

Footnotes

↵* This work was supported by Grants HL30657 (to J. E. B. F.), DK38452 (to J. H. H.), and HL56252 (to J. H. H.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ABP-280

actin-binding protein-280

GP

glycoprotein

GAL

galactosidase

kb

kilobase(s)

aa

amino acid(s)

MAb

monoclonal antibody

GST

glutathione S-transferase

FcγRI

IgG Fc receptor I.
↵² G. Liu, L. Thomas, C. C. Cunningham, J. H. Hartwig, and G. Thomas, manuscript in preparation.
↵³ C. Kedinger, personal communication.
- Received September 9, 1996.
- Revision received November 8, 1996.