Fig. 6.

Effect of CysC95-146 and GPR15L on GPR15 function. (A) GPR15L does not inhibit SIVmac239 infection. GHOST-GPR15 cells were preincubated with increasing amounts of GPR15L or CysC95-146, infected with an SIVmac239 luciferase reporter virus, and infection rates determined 3 d later. Experiments shown in all panels were performed at least in triplicate and curves show mean values (±SEM). (B and C) GPR15L (B) but not CysC95-146 (C) down-modulate GPR15 from the surface of GHOST-GPR15 and CEM-M7 cells. GHOST-GPR15 or CEM-M7 cells were preincubated with increasing amounts of GPR15L or CysC95-146 at 4 °C. GPR15 expression was analyzed by flow cytometry. (D) Cystatin C fragments do not induce GPR15-mediated calcium signaling. The effect of the indicated CysC fragments and GPR15L on calcium signaling was detected by measuring aequorin fluorescence in GPR15-expressing CHO-K1 cells. Each data point represents the mean relative light units ± SD over background fluorescence of quadruplicate measurements. (E) Cystatin C fragments do not interfere with the signaling function of GPR15L. The effect of CysC fragments on GPR15L signaling was analyzed as described in D. (F) GPR15L does not enhance the antiviral activity of CysC95-146. GHOST-GPR15 cells were treated with CysC95-146 in the presence or absence of GPR15L and infected with a SIVmac239 luciferase reporter virus as described in A. (G) CysC95-146 competes with antibodies targeting the N terminus or ECL1 region for GPR15 binding. GHOST-GPR15 cells were incubated with the indicated concentrations of CysC95-146 and either ab8104 targeting the N terminus of GPR15 or ab188938 targeting the first ECL of GPR15, washed, stained with a PE-conjugated secondary antibody, and analyzed by flow cytometry. The indicated frequencies of GPR15-positive cells were obtained by subtraction of background (secondary antibody only), followed by normalization to the no-peptide samples (100%). Shown are the mean values for n = 3 experiments ± SEM, **P < 0.01 (Welch’s t test, unpaired). (H) Anti-GPR15 antibodies ab8104 targeting the N terminus and ab188938 targeting the first extracellular loop of GPR15 but not RD #367902 (unknown binding site) inhibit SIVmac infection. GHOST-GPR15 cells were preincubated with the indicated anti-GPR15 antibodies prior to infection with a SIVmac239 luciferase reporter virus. (I) Theoretically proposed binding of CysC95-146 with GPR15 based on reactive molecular dynamics (MD) simulations (ReaxFF). In the model, the amino acid-resolved interaction energies are highlighted using the color coding shown in the Lower Right scale.