abstract
- The formyl peptide receptor (FPR) has been widely used to study the kinetics of the interaction between ligand, receptor and G protein with real-time fluorescence methods. Because the wild type receptor rapidly signals, and is then desensitized and internalized once occupied by ligand, it has been difficult to study the uncoupled receptor form. We have examined a mutant form of the FPR expressed in U937 cells that does not bind G protein and is thus ideal to study the uncoupled form of the FPR in the intact cell. Using kinetic flow cytometry, we have measured the dissociation kinetics of a fluorescent ligand from this mutant in intact, permeabilized and fixed cells. We observed a novel uncoupled receptor form in the intact cell with a dramatically reduced off-rate (approximately 0.02 s-1) from LR in a broken cell preparation (approximately 0.2 s-1). Both receptor forms are retained in the presence of formaldehyde. We also observed this novel receptor form coexisting with the LRG complex when the wild type receptor is fixed in neutrophils or transfectants. These results complex when the wild type receptor is fixed in neutrophils o transfectants. These results lead us to suggest that there are distinct receptor structures in cells and membranes and that only a fraction of receptors in intact cells exist in the uncoupled form.