The Transmembrane Domain of Glycoprotein Ibβ Is Critical to Efficient Expression of Glycoprotein Ib-IX Complex in the Plasma Membrane

José A. López,Arnoldo Padilla,Xi Mo,Nan Lu,Renhao Li

doi:10.1074/jbc.m600924200

José A. López, Arnoldo Padilla + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m600924200

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Abstract

Lack of expression of glycoprotein (GP) Ib-IX-V complex in platelets often results from mutations in its three subunits: GP Ibalpha, GP Ibbeta, or GP IX. The requirement of all three subunits in the efficient surface expression of the receptor complex has been reproduced in Chinese hamster ovary cells. Here, we probed the role of the transmembrane domains in expression of the GP Ib-IX complex and potential interactions between these domains. Replacing the transmembrane domains of either GP Ibalpha or GP Ibbeta, but not that of GP IX, with unrelated sequences markedly diminished surface expression of the GP Ib-IX complex in transiently transfected Chinese hamster ovary cells. Replacement of the Ibbeta transmembrane domain produced the largest effect. Furthermore, several single-site mutations in the Ibbeta transmembrane domain were found to significantly decrease overall expression as well as surface expression of GP Ibalpha, probably by perturbing the interaction between the Ibalpha and Ibbeta transmembrane domains and in turn reducing the stability of GP Ibalpha in the cell. Mutations S503V and S503L in the Ibalpha transmembrane domain partly reversed the expression-decreasing effect of mutation H139L, but not the others, in the Ibbeta transmembrane domain, suggesting a specific interaction between these two polar residues. Together, our results have demonstrated the importance of the Ibbeta transmembrane domain, through its interaction with the Ibalpha counterpart, to the proper assembly and efficient surface expression of the GP Ib-IX complex.

Highlights

The GP Ib-IX-V complex is widely considered to comprise GP Ib␣, GP Ib␤, GP IX, and GP V with a 2:2:2:1 stoichiometry [5,6,7], it is not clear how these subunits assemble into a functional receptor complex in the membrane, partly because intersubunit interactions are largely unknown
We have identified single-site mutations in the Ib␤ TM domain that significantly decrease the complex expression by affecting the stability of GP Ib␣, suggesting that these mutations affect the interaction between GP Ib␤ and Ib␣
Since this study addresses mostly the mutational effects on surface expression of the GP Ib-IX complex in transfected Chinese hamster ovary (CHO) cells, it is critical that changes in expression be attributed only to the mutations

Summary

Introduction

The GP Ib-IX-V complex is widely considered to comprise GP Ib␣, GP Ib␤, GP IX, and GP V with a 2:2:2:1 stoichiometry [5,6,7], it is not clear how these subunits assemble into a functional receptor complex in the membrane, partly because intersubunit interactions are largely unknown. We found that replacing the Ib␤ TM domain with unrelated sequences produced the largest effect, in comparison with TM replacements in GP Ib␣ and GP IX, on surface expression of the GP Ib-IX complex in transfected CHO cells.

Results

Conclusion