Inositol phosphate (IP) accumulation assay

Flag-tagged wild-type and mutant CCR5 receptors were cloned into the expression vector pTT5 (Invitrogen) and expressed in HEK293F cells along with the chimeric Gα protein (Gα_Δ6qi4myr) at the ratio of plasmids of 2:1 (w/w). Cells were harvested 48 h posttransfection. To measure the cell-surface expression of CCR5, 10 μl cells were mixed with 15 μl Monoclonal ANTI-FLAG M2-FITC antibody (Sigma, F4049; 1:100 diluted by TBS supplemented with 4% BSA). After 20 min reaction, the fluorescence signal on the cell surface was measured by an FCM (flow cytometry) reader (Millipore).

IP1 accumulation was measured using an IP-One Gq assay kit (Cisbio Bioassays, 62IPAPEB) following the manufacturer’s instructions. In brief, the harvested cells were plated in 384-well plates (30,000 cells per well) and treated with different concentrations of MIP-1α or RANTES (1 pM–10 μM diluted in stimulation buffer; see below for expression and purification of the chemokines) at 37 °C for 90 min. Then 3 μl cryptate-labeled anti-IP1 monoclonal antibody and 3 μl d2-labeled IP1, which were pre-diluted in Lysis Buffer (1:20), were added to the wells, and incubated at room temperature for 1 h. The plates were then read by an EnVision multilabel plate reader (PerkinElmer) with excitation at 330 nm and emission at 620 and 665 nm. The values were then converted to IP production by a standard dose-response curve using GraphPad Prism 8.0 (GraphPad Software). The assays were performed in parallel with the measurement of the IP production using the cells only transfected with the receptor as a control. The Gα_Δ6qi4myr-mediated IP accumulation was calculated by subtracting the portion of control-mediated IP production for the wild-type receptor and all the mutants. EC₅₀ and pEC₅₀ ± SEM were calculated using nonlinear regression (curve fit) in GraphPad Prism 8.0.