Background: The mechanism of assembly of the platelet glycoprotein (GP) Ib-IX complex from GPIbalpha, GPIbbeta and GPIX subunits is not entirely clear. In this complex, ectodomains of both GPIbbeta and GPIX subunits contain two leucine-rich repeats (LRR) and share high sequence similarity. However, they differ noticeably in stability, hampering further analysis of their interaction.
Objectives and methods: Guided by analysis of the LRR structure, we report a well-folded Ibbeta/IX chimera and its usage in dissecting GPIX function.
Results: In this chimera, three non-contiguous sequences that may constitute the putative convex surface of the GPIbbeta ectodomain are replaced by their GPIX counterparts. Like GPIbbeta but unlike GPIX ectodomain, it can secrete from transfected Chinese hamster ovary cells and fold into a stable conformation. Furthermore, replacing the ectodomain in GPIX with the Ibbeta/IX chimera, but not the GPIbbeta ectodomain, preserved its interaction with GPIbbeta as demonstrated by its native-like GPIbbeta-induced increase in surface expression and coimmunoprecipitation.
Conclusions: The putative convex surface of the LRR domain in GPIX is sufficient, in the context of full-length subunit, to mediate its association with GPIbbeta.