Specific Heteromeric Association of Four Transmembrane Peptides Derived from Platelet Glycoprotein Ib–IX Complex

详细信息    查看全文

• 作者：Shi-Zhong Luo ; Renhao Li
• 关键词：GP Ib– ; IX complex ; transmembrane domain interaction ; complex assembly ; thiol– ; disulfide exchange ; fluorescence resonance energy transfer
• 刊名：Journal of Molecular Biology
• 出版年：2008
• 出版时间：3 October 2008
• 年：2008
• 卷：382
• 期：2
• 页码：448-457
• 全文大小：744 K

文摘

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 Ibα, GP Ibβ 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 Ibβ TM sequence, but not the Ibα and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ibβ–Ibα–Ibβ peptide complex (αβ₂) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibα and Ibβ TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native αβ₂ 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 αβ₂ 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 αβ₂ 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 Ibα, Ibβ 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.