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Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha.

ABSTRACT: Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIb?, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIb? sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIb?, GPIb?, and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIb? is critical for ADAM17-dependent shedding of GPIb? induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIb? and GPIb? subunits further lessened the restraint of GPIb? on GPIb? shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIb? cytoplasmic domain disrupted calmodulin binding to GPIb? but had little effect on GPIb? shedding. Analysis of induction of GPIb? shedding by membrane-permeable GPIb?-derived peptides implicated the association of GPIb? with an unidentified intracellular protein in mediating regulation of GPIb? shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.

SUBMITTER: Mo X

PROVIDER: S-EPMC2952211 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha.

Mo Xi X Nguyen Nam X NX Mu Fi-Tjen FT Yang Wenjun W Luo Shi-Zhong SZ Fan Huizhou H Andrews Robert K RK Berndt Michael C MC Li Renhao R

The Journal of biological chemistry 20100817 42

Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIbα, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIbα sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIb ...[more]

PMID: 20716526

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Project description:Binding of platelet receptor glycoprotein Ibα (GPIbα) to the A1 domain of von Willebrand factor (vWF) is a critical step in both physiologic hemostasis and pathologic thrombosis, for initiating platelet adhesion to subendothelium of blood vessels at sites of vascular injury. Gain-of-function mutations in GPIbα contribute to an abnormally high-affinity binding of platelets to vWF and can lead to thrombosis, an accurate complication causing heart attack and stroke. Of various antithrombotic monoclonal antibodies (mAbs) targeting human GPIbα, 6B4 is a potent one to inhibit the interaction between GPIbα and vWF-A1 under static and flow conditions. Mapping paratope to epitope with mutagenesis experiments, a traditional route in researches of these antithrombotic mAbs, is usually expensive and time-consuming. Here, we suggested a novel computational procedure, which combines with homology modeling, rigid body docking, free and steered molecular dynamics (MD) simulations, to identify key paratope residues on 6B4 and their partners on GPIbα, with hypothesis that the stable hydrogen bonds and salt bridges are the important linkers between paratope and epitope residues. Based on a best constructed model of 6B4 bound with GPIbα, the survival ratios and rupture times of all detected hydrogen bonds and salt bridges in binding site were examined via free and steered MD simulations and regarded as indices of thermal and mechanical stabilizations of the bonds, respectively. Five principal paratope residues with their partners were predicted with their high survival ratios and/or long rupture times of involved hydrogen bonds, or with their hydrogen bond stabilization indices ranked in top 5. Exciting, the present results were in good agreement with previous mutagenesis experiment data, meaning a wide application prospect of our novel computational procedure on researches of molecular of basis of ligand-receptor interactions, various antithrombotic mAbs and other antibodies as well as theoretically design of biomolecular drugs.

Dataset Information

Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha.

Publications

Transmembrane and trans-subunit regulation of ectodomain shedding of platelet glycoprotein Ibalpha.

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