Fig. 8. CysLT₁ receptor expression and heterologous desensitization in COS-7 cells. Confocal microscopy of the CysLT₁ receptor trafficking in intact COS-7 cells. (A) COS-7 cells expressing HA-tagged CysLT₁ receptor were immunoprecipitated with HA monoclonal antibody and visualized with an CysLT₁ polyclonal antibody. (B) Equilibrium mixed-type binding curve of [³H]LTD₄ in membranes from COS-7 cells transiently transfected with HA-tagged CysLT₁ receptor. Binding of [³H]LTD₄ is expressed as the ratio of bound ligand concentration to total ligand concentration (B/T, dimensionless) versus the logarithm of total ligand concentration (Log T). Two independent [³H]LTD₄ mixed-type experiments were performed, each with triplicate determinations, and analyzed simultaneously. For the sake of clarity, only one representative curve is shown; the curve is computer generated. (Inset) The percentage of total [³H]LTD₄ binding and displacement by unlabeled LTD₄, ATP or UDP. (C) Representative traces of the [Ca²⁺]_i transient induced by 30 µM ATP (2nd challenge) before and after an initial challenge with 1 µM LTD₄. (D) Representative traces of the [Ca²⁺]_i transient induced by 1 µM LTD₄ (2nd challenge) before and after an initial challenge with 30 µM of ATP. (E) Confocal laser scanning microscopy of the subcellular distribution of the CysLT₁ receptors performed using a specific antibody raised against the C-terminal tail. Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 in basal conditions (control) and after a 5 minute challenge with the indicated stimuli. Immunocytochemical staining of COS-7 cells after stimulation with vehicle (left), 1 µM LTD₄ (middle), 30 µM ATP (right). The experiment shown is representative of two others performed.