Abstract:
:As the receptor on the platelet surface for von Willebrand factor, glycoprotein (GP) Ib-IX complex is critically involved in hemostasis and thrombosis. How the complex is assembled from GP Ibalpha, GP Ib beta and GP IX subunits, all of which are type I transmembrane proteins, is not entirely clear. Genetic and mutational analyses have identified the transmembrane (TM) domains of these subunits as active participants in assembly of the complex. In this study, peptides containing the transmembrane domain of each subunit have been produced and their interaction with one another characterized. Only the Ib beta TM sequence, but not the Ibalpha and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ib beta-Ibalpha-Ib beta peptide complex (alphabeta(2)) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibalpha and Ib beta TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native alphabeta(2) complex, reproducing the same effect by the IX subunit in cells expressing the GP Ib-IX complex. Specific fluorescence resonance energy transfer was observed between donor-labeled alphabeta(2) peptide complex and acceptor-conjugated IX TM peptide in micelles. Finally, the mutation D135K in the IX TM peptide could hamper both the formation of the alphabeta(2) complex and the energy transfer, consistent with its reported effect in the full-length complex. Overall, our results have demonstrated directly the native-like heteromeric interaction among the isolated Ibalpha, Ib beta and IX TM peptides, which provides support for the four-helix bundle model of the TM domains in the GP Ib-IX complex and paves the way for further structural analysis. The methods developed in this study may be applicable to other studies of heteromeric interaction among multiple TM helices.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Luo SZ,Li Rdoi
10.1016/j.jmb.2008.07.037subject
Has Abstractpub_date
2008-10-03 00:00:00pages
448-57issue
2eissn
0022-2836issn
1089-8638pii
S0022-2836(08)00879-6journal_volume
382pub_type
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