Abstract
Platelet glycoprotein (GP) Ib-IX-V complex is critically involved in the early steps of platelet adhesion and activation at sites of vessel wall injury. A functional GP Ib-IX complex comprising GP Ibα, GP Ibβ, and GP IX can be expressed in transfected cells, with efficient expression on the cell surface requiring all of its three subunits. As a first step to elucidate the 3-dimensional organizing principle of this receptor complex, we probed the interactions of its transmembrane (TM) domains. Replacement of the TM domains of either GP Ibα or GP Ibβ with poly-Leu or poly-Leu-Ala sequences markedly diminished cell surface expression of the complex, as assessed by flow cytometry and western blotting of transiently transfected CHO cells. Similar replacements of the GP IX TM domain had little effect on complex expression. TM replacements in GP Ibα or GP Ibβ also affected formation of the membrane-proximal inter-subunit disulfide bond between the two subunits. A thiol-disulfide exchange assay with the TM-containing peptides dispersed in dodecylphosphocholine micelles demonstrated a direct and specific interaction between the Ibα and Ibβ TM peptides, as the peptides preferentially formed a disulfide-linked αβ hetero-dimer rather than αα or ββ homo-dimers. Replacement mutations in the Ibα TM peptide abolished formation of the hetero-dimer. Further mutagenesis studies produced several single-site mutations within the TM domains of GP Ibα and GP Ibβ that drastically lowered the level of surface expression of the GP Ib-IX complex. These mutations also disrupted formation of the inter-subunit disulfide bond in the transfected GP Ib-IX complex as well as in the context of isolated TM peptides. These results demonstrate a specific interaction between the TM domains of GP Ibα and GP Ibβ, which can be abolished by whole-domain replacement or site-specific mutations, significantly diminishing assembly and cell-surface expression of the GP Ib-IX complex.
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