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Fig. 6. PKA phosphorylation is important for nuclear translocation of ß-arrestin2. (A) Treatment with PKA inhibitor H-89 blocks ß-arrestin2 redistribution in human sperm in response to bourgeonal treatment. Human sperm were pretreated with different concentrations of H-89 inhibitor as described and stimulated with bourgeonal for 20 minutes. Cells still showing control-like mid-piece staining with the anti-ß-arrestin2 antibody after bourgeonal stimulation were counted and plotted against the H-89 concentration. (B) Site-directed mutagenesis experiments were performed to delete the potential PKA phosphorylation sites within the third intracellular loop (positions S230A, S232A, S239A; PM^*3-loop) and the C-terminus (position T312A; PM^*C-term) of hOR17-4 as described in the Materials and Methods. Both mutant receptors failed to induce nuclear translocation of ß-arrestin2-GFP in HEK293 cells upon bourgeonal treatment. A similar failure to induce nuclear translocation of ß-arrestin2 was observed with the wild-type receptor after inhibition of PKA with H-89. At least 5000 cells were investigated; the number of cells showing nuclear ß-arrestin2 accumulation was counted and given as percentage of total cells investigated. Error bars represent s.d. (^**P<0.01 compared with untreated cells). ND, no cells with nuclear ß-arr2 detected. (C) Mutation of the potential PKA phosphorylation sites leads to the increased response duration in Ca²⁺-imaging experiments in transfected HEK293 cells. Duration of the Ca²⁺ rise was measured from the onset of the stimulus to the return to basal levels.

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