Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX complex that binds to purified actin-binding protein.
The platelet membrane glycoprotein (GP) Ib-IX complex is a major site of attachment of the platelet membrane skeleton to the plasma membrane. This association is mediated by the interaction of actin-binding protein with the GP Ib-IX complex. The aim of the present work was to identify domains on the GP Ib-IX complex that interact with actin-binding protein. Synthetic peptides corresponding to sequences of the GP Ib alpha-chain and beta-chain cytoplasmic domains were analyzed for their ability to bind to purified actin-binding protein. Two overlapping peptides encompassing a sequence (Thr-536-Phe-568) from the central region of the cytoplasmic domain of GP Ib alpha were the most effective in binding 125I-actin-binding protein, as assessed by a microtiter well approach and peptide affinity chromatography. One of the active peptides (Thr-536-Leu-554) was chosen to evaluate the likelihood that the central region of the cytoplasmic domain of GP Ib alpha is involved in binding of the intact complex to actin-binding protein. This peptide could be specifically cross-linked to purified actin-binding protein in solution. Rabbit polyclonal antibody against this peptide inhibited the binding of purified actin-binding protein to the purified GP Ib-IX complex. Finally, as in intact platelets, the calpain-induced hydrolytic fragments of purified actin-binding protein (M(r) = 200,000 and M(r) = 91,000) showed little binding to the GP Ib alpha peptide. Taken together, these results provided evidence that a region between Thr-536 and Phe-568 of the cytoplasmic domain of GP Ib alpha participates in the interaction of the GP Ib-IX complex with actin-binding protein.
Andrews
R K
RK
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94141-9100.
Fox
J E
JE
D000595:Amino Acid Sequence; D001345:Autoradiography; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism
D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D010980:Platelet Membrane Glycoproteins