The GPI anchor of tetherin/BST-2 is needed to regulate P2Y₁₂R function. (A) An overlay of the P2Y₁₂R crystal structures: antagonist bound (wheat, 4NTJ)^x28  and agonist bound (cyan,4PXZ)^xx.²⁹  Protein structures are shown as ribbons, and cholesterol (wheat) and 1-oleoyl-R-glycerol (cyan) are shown in space-filling representations. TM-helices binding these lipids are labeled I, VI, and VII. (B) A model of the tetherin/BST-2-GPI interaction with P2Y₁₂R. C-terminal part of tetherin/BST-2, magenta ribbon; GPI anchor in space-filling representations with the lipid tails in the membrane packed against agonist-bound P2Y₁₂R, shown as cyan ribbon. Molecular modeling showing potential interaction between tetherin/BST-2 and the P2Y₁₂R. (C) The GPI anchor of tetherin/BST-2 is necessary for P2Y₁₂R interaction. HEK293 cells were transfected with FLAG-P2Y₁₂ alone or in combination with a variety of HA-tagged constructs, including full-length tetherin/BST-2, 2 truncations lacking either the N-terminal region (ΔN-HA) or the GPI anchor (ΔGPI-HA) of tetherin/BST-2, tetherin/BST-2 triple cysteine→alanine mutant (C3A-HA), or a tetherin/BST-CD8 construct in which the GPI anchor has been replaced by the CD8α transmembrane domain (ΔGPI-CD8-HA). Cells were lysed, and the receptor was immunoprecipitated by using monoclonal mouse anti-FLAG M2 Affinity Gel. Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted for associated anti-HA (top gel). Inputs were immunoblotted for anti-HA to show protein loading (middle gel) and immunoprecipitates reprobed with anti-FLAG antibody (rabbit) to show the total receptor immunoprecipitated (bottom gel). (D) The GPI anchor of tetherin/BST-2 is essential for tetherin/BST-2–dependent regulation of P2Y₁₂R activity. HEK293 cells were cotransfected with 2:1:1 ratios of G_i protein constructs (Rluc-II-tagged Gαi-1/Gβ1/GFP10-tagged Gγ2), FLAG-P2Y₁₂R and either HA-tagged tetherin/BST-2 constructs (HA-WT, ΔGPI-HA or CD8-ΔGPI-HA) or pcDNA3.1 control. P2Y₁₂R activity was assessed by agonist-stimulated changes in BRET between Gαi-1-Rluc-II and GFP10-Gγ2 in living cells. Data are expressed as ΔBRET by subtracting the BRET values obtained in the vehicle condition from the one measured with ADP and represent mean ± standard error of the mean of 4 independent experiments. Shown are concentration-dependence of P2Y₁₂R activation (ADP; 1 nM-100 µM; top) and time-dependent activation of the P2Y₁₂R by ADP (20 µM; bottom). Only full-length tetherin/BST-2-HA expression attenuated P2Y₁₂R activity (both top and bottom; *P < .05; 2-way ANOVA tetherin/BST-2 vs pDNA3.1 control). (E) Tetherin/BST-2 attenuatedP2Y₁₂R mobility in membrane microdomains after receptor activation, as assessed by FRAP. HEK293 cells expressing mCherry-HA-P2Y₁₂R alone or coexpressing mCherry-P2Y12-HA and BST-2-GFP, were stained with the membrane microdomain marker cholera toxin-B (CTB). Confocal FRAP was performed at 37°C in live cells with a 2-μm diameter bleach spot (supplemental Figure 4B) on CTB⁺ (yellow circles) or negative regions (cyan circles) of the cell membrane. FRAP was subsequently assessed. In agonist-treated cells ADP (20 µM) was added 5 minutes before FRAP. Data were collected from >9 cells from 3 independent experiments and are expressed as either the percentage of normalized mCherry-P2Y12-HA final mobile fraction (left) or the diffusion coefficient (mm²/s; right). Tetherin/BST-2 expression significantly attenuated P2Y₁₂R mobility into CTB⁺ membrane microdomain regions in ADP-treated cells (left; *P < .05 Mann-Whitney U test in tetherin/BST-2 expressing vs nonexpressing cells in membrane microdomains in ADP-treated cells (right); *P < .05; Student t test; tetherin/BST-2–expressing vs nonexpressing cells in membrane microdomains in ADP-treated cells).