Transmembrane domains are critical to the interaction between platelet glycoprotein V and glycoprotein Ib-IX complex

Abstract

Background: The glycoprotein (GP) Ib-IX-V complex, the von Willebrand factor receptor on the platelet surface, is critically involved in hemostasis and thrombosis. The GPV subunit interacts with GPIb-IX to form the GPIb-IX-V complex, but the underlying molecular basis remains unclear. It was observed earlier that efficient expression of GPV in the plasma membrane requires co-expression of GPIb-IX.

Objectives and methods: Hypothesizing that GPIb-IX stabilizes GPV through direct interaction and consequently enhances GPV surface expression, we aim in this study to identify structural elements in the complex that mediate the interaction between GPV and GPIb-IX by analyzing mutational effects on GPV surface expression in transfected Chinese hamster ovary cells.

Results: Enhancement of GPV surface expression by GPIb-IX requires transmembrane domains of both GPV and GPIbα, as replacing the GPV transmembrane domain with an unrelated poly-leucine-alanine sequence abolished the enhancing effect of GPIb-IX. Additional mutagenesis analysis of the GPV transmembrane helix identified three helical sides containing conserved polar residues as critical to efficient GPV surface expression. Similarly, replacing residues in three sides (Gly495/Ala502/Leu509, Phe491/Trp498/Val505, and Y492/L499/L506) of the GPIbα transmembrane domain with leucines preserved the surface expression level of GPIb-IX but significantly altered that of GPV.

Conclusions: Our results demonstrate for the first time the importance of transmembrane domains for efficient surface expression of GPV and suggest that GPV and GPIbα transmembrane domains interact with each other, contributing to assembly of the GPIb-IX-V complex.

Figure 1

Efficient surface expression of GPV depends on the presence of GPIb-IX. CHO K1 cells were transiently transfected with GPV cDNA in the absence or presence of GPIb-IX genes. Two days after transfection, surface expression levels of GPIbα, GPIX and GPV were detected by flow cytometry with antibodies WM23, FMC25 and SW16, respectively. (A) Overlaid flow cytometry histograms from the same transfection experiment comparing the surface expression levels of GPIbα, GPIX and GPV in various transfected CHO cells. Identities of transfected cells are denoted by the transfected subunits. The overlaid histograms are representative of 3–5 independent experiments. (B) Bar plots of surface expression levels of GPIbα (open bar), GPIX (slashed bar) and GPV (black bar), quantified as relative mean fluorescence intensity (MFI). The fluorescence intensity measured from 10,000 cells was normalized to the value in CHO-αβIXV cells as 100% and that in CHO-vector cells 0% (not shown). The data are presented as the mean ± S.D. calculated from 3–5 independent experiments. Groups were compared using the non-paired t test; **, p < 0.001.

Figure 2

Replacing the GPV TM domain with an unrelated sequence reduces its surface expression in the presence of GPIb-IX. (A) Sequences of wild-type or poly-Leu-Ala-containing GPV TM domains. The GPV TM domain sequence, marked by the starting and ending residue numbers, was replaced by a poly-Leu-Ala sequence in GPV_pLA. The extracellular and cytoplasmic domains remained unchanged in GPV_pLA. (B, C) Overlaid histograms and quantitative bar plots showing the surface expression level of the TM-replaced GPIb-IX-V complex. GPV and GPV_pLA was each transfected into CHO cells in the absence or presence of GPIb-IX, and surface expression levels of GPIbα, GPIX and GPV were detected by flow cytometry with antibodies WM23, FMC25 and SW16, respectively. The legends follow that used in Figure 1. The data are presented as the mean ± S.D. calculated from 3–5 independent experiments. Groups were compared using the non-paired t test; **, p < 0.001.

Figure 3

Side-scanning mutagenesis of the GPV TM domain. (A) The helical wheel view of the GPV TM domain. Residues in the TM domain were divided into seven sides, each of which was assigned from a to g. The sides identified as important are boxed in green. (B) TM sequences of the wild-type (GPV) and seven side-scanning mutants (GPV_a - GPV_g). Residue numbers are on the top. The mutations in each sequence are underlined. (C) Bar plot of the surface expression levels of GPIbα (open bar), GPIX (slashed bar) and GPV (black bar), quantified as relative MFI, in side-scanning mutant cells. The data are presented as the mean ± S.D. from 3–6 independent experiments. Groups were compared using the non-paired t test; *, p < 0.05; **, p < 0.001.

Figure 4

The TM domain of GPV is important for its surface expression in lentiviral-transfected CHO cells. The lentivirus containing a copy of GPV gene or empty lentivirus was used to infect CHO K1 cells or CHO cells stably expressing GPIb-IX (stable CHO-αβIX cells). After infected cells were cultured for 4–7 days, surface expression levels of GPIbα, GPIX and GPV were detected by flow cytometry with antibodies WM23, FMC25 and SW16, respectively. (A) Overlaid flow cytometry histograms comparing the surface expression levels of GPIbα (left panel) and GPIX (right panel) in stable CHO-αβIX cells before (black trace) and after (blue) GPV-containing lentivirus infection. The grey-filled histograms were obtained using CHO K1 cells before infection. The histograms are representative of 2 independent experiments. (B) Overlaid flow cytometry histograms comparing surface expression levels of GPV (black trace) and GPV_pLA (red) in lentiviral-transfected CHO K1 cells (left panel) or stable CHO-αβIX cells (right panel). The grey-filled histograms were obtained using the empty lentivirus. The histograms are representative of 3–4 independent experiments. (C) Bar plots of surface expression levels of GPV and its TM mutants as illustrated in Figure 2A and 3B, quantified as relative MFI, in lentiviral-transfected CHO-αβIX cells (white bar) and CHO K1 cells (grey bar). The data are presented as the mean ± S.D. from 2–4 independent experiments. Groups were compared using the non-paired t test; *, p < 0.01.

Figure 5

The polar residues in the GPV TM domain are critical to its interaction with GPIb-IX. (A) TM sequences of the wild-type GPV and polar-residue mutants, with the subscript describing mutations within. In each sequence, the actual mutations are underlined. (B) Bar plot of surface expression levels of GPV polar mutants when they are expressed alone in transfected cells. The level is measured by flow cytometry, quantified and expressed as a percentage of that in CHO-αβIXV cells. (C) Bar plot of the surface expression levels of GPIbα (open bar), GPIX (slashed bar) and GPV (black bar), quantified as relative MFI, in transfected cells expressing GPV polar mutants. The data are presented as the mean ± S.D. from 3–4 independent experiments. Groups were compared using the non-paired t test; *, p < 0.05.

Figure 6

Side-scanning mutagenesis of the GPIbα TM domain. (A) The helical wheel view of the GPIbα TM domain. Residues in the TM domain were divided into seven sides, each of which was assigned from a to g. The sides identified as important are boxed in magenta. (B) TM sequences of the wild-type (GPIbα) and seven side-scanning mutants (GPIbα_a - GPIbα_g). Residue numbers are on the top. The mutations in each sequence are underlined. (C) Overlaid flow cytometry histograms showing surface expression levels of GPIbα in transiently transfected CHO cells expressing various GPIbα TM mutants. The wild-type trace is in black, and the seven mutants are rainbow colored as indicated in (B). The overlaid histograms are representative of 3–4 independent experiments. (D) Bar plot of the surface expression levels of GPIbα (open bar) and GPIX (slashed bar), quantified as relative MFI, in cells transfected with GPIb-IX genes. (E) Bar plot of the surface expression levels of GPIbα (open bar), GPIX (slashed bar) and GPV (black bar), quantified as relative MFI, in transfected with GPIb-IX-V genes. The data are presented as the mean ± S.D. from 3–5 independent experiments. Groups were compared using the non-paired t test; *, p < 0.01; **, p < 0.001.

Figure 7

The GPIbα TM domain is accessible to GPV association. (A) A ribbon diagram showing a structural model of GPIb-IX complex, adapted from a recent report [13]. The model contains the membrane-proximal stalk region and TM domain of GPIbα (magenta), extracellular and TM domains of GPIbβ (light and dark blue) and GPIX (orange). The membrane-distal portion of GPIbα extracellular domain and all the cytoplasmic domains are not shown. (B) The same model was rotated by 90 degrees horizontally, showing the accessibility of GPIbα TM domain to direct association of GPV TM domain (green) as well as the inaccessibility of GPIX TM domain.