The cytoplasmic domain of the alpha-subunit of glycoprotein (GP) Ib mediates attachment of the entire GP Ib-IX complex to the cytoskeleton and regulates von Willebrand factor-induced changes in cell morphology.
The glycoprotein (GP) Ib-IX complex is one of the major platelet membrane glycoproteins.  Its extracellular domain binds von Willebrand factor at a site of injury, an interaction that leads to activation of intracellular pathways.  Its intracellular domain associates tightly with the platelet cytoskeleton through actin-binding protein.  The goal of the present study was to investigate the role of the cytoplasmic domain of the GP Ib-IX complex and its interaction with the cytoskeleton.  Cultured cells were transfected with the cDNAs coding for GP Ib(beta), GP IX, and full-length or truncated forms of GP Ib(alpha). Western blots of detergent-insoluble fractions of Triton X-100-lysed cells showed that deletion of amino acids Trp-570 to Ser-590 from the cytoplasmic domain of GP IB(alpha) abolished the interaction of the entire GP Ib-IX complex with the cytoskeleton.  Truncated GP Ib(alpha) that was unable to associate with the cytoskeleton retained its ability to associate with GP Ib(beta), to be inserted into the membrane, and to bind von Willebrand factor.  Cells expressing GP Ib(alpha) changed their shape following adhesion to immobilized von Willebrand factor.  Cells expressing truncated GP Ib(alpha) also changed their shape following adhesion but showed a very different morphology as compared to cells expressing full-length GP Ib(alpha).  These results show that GP Ib-IX-von Willebrand factor interactions lead to cytoskeletal reorganizations, that the cytoplasmic domain of GP Ib(alpha) regulates these reorganizations, and that the cytoplasmic domain of GP Ib(alpha) is absolutely required for attachment of the GP Ib-IX complex to the cytoskeleton.
Cunningham
J G
JG
Children's Hospital Oakland Research Institute, California 94609, USA.
Meyer
S C
SC
Fox
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D006224:Cricetinae; D003435:Crotalid Venoms / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D017931:DNA Primers; D006388:Hemagglutinins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008545:Melanoma; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014841:von Willebrand Factor / Q000378:metabolism* / Q000494:pharmacology*
D003435:Crotalid Venoms; D017931:DNA Primers; D006388:Hemagglutinins; D046911:Macromolecular Substances; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C030961:botrocetin