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First published online 4 July 2006
doi: 10.1242/jcs.03046

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Novel function of ß-arrestin2 in the nucleus of mature spermatozoa

Eva M. Neuhaus^1,*,, Anastasia Mashukova^1,*, Jon Barbour¹, Dirk Wolters² and Hanns Hatt¹

¹ Department of Cell Physiology, Ruhr-Universitaet Bochum, Universitaetsstr. 150, 44780 Bochum, Germany
² Department of Analytical Chemistry, Ruhr-Universitaet Bochum, Universitaetsstr. 150, 44780 Bochum, Germany

View larger version (44K): [in a new window]   Fig. 1. ß-arrestin2 accumulates in the nucleus and acquires a modulatory function in a GAL4-based transcription assay upon odorant receptor stimulation. (A) Confocal microscopy of HEK293 cells cotransfected with hOR17-4 and ß-arrestin2-GFP before (0) and after (20 min) bourgeonal stimulation. Bourgeonal treatment causes nuclear translocation of ß-arrestin2-GFP. (B) Quantification of the fluorescence intensities in the nucleus relative to intensities in the cytosol measured in single confocal sections. The averages of at least 100 HEK293 cells demonstrate a fourfold increase in the amount of nuclear ß-arrestin2 in responding cells. Error bars represent s.d. (**P<0.01 compared with levels in the control). (C) Pre-treatment of cells with concanavalin A (+conA) as inhibitor of clathrin-mediated endocytosis blocks ß-arrestin2-GFP nuclear translocation, pre-treatment with ß-cyclodextrin (+CD) as inhibitor of caveolae-mediated endocytosis has no effect. At least 5000 cells were investigated and the number of cells showing nuclear accumulation of ß-arrestin2 was counted and given as percentage of the total number of cells investigated. Error bars represent SD (**P<0.01). (D) Nuclear translocation of ß-arrestin2-GFP is specific to hOR17-4 activation. The upper panel shows changes in the Ca2+ concentration in Calcium-Orange-loaded individual cells that were cotransfected with hOR17-4 and ß-arrestin2-GFP in pseudocolors; the lower panel shows ß-arrestin2-GFP localization. Bourgeonal (5 µM) was present during the whole experiment. The ß-arrestin2-GFP expressing cell which show nuclear translocation of the ß-arrestin2 also showed an increase in the intracellular Ca2+ concentration in response bourgeonal (5 µm). The curve shows the mean change in the fluorescence intensities in response to Borgeonal application as a function of time for ten Calcium Orange loaded cells cotransfected with ß-arrestin2-GFP and hOR17-4. (E) Western blot analysis of fractionated HEK293 cells cotransfected with ß-arrestin2 and hOR17-4, nonstimulated (-) and stimulated (+) with bourgeonal for 20 minutes. The purity of the nuclear fraction was verified by staining with anti-ß-actin antibodies. Nuclear ß-arrestin2 signals were quantified by densitometry; the graph depicts the average from three independent experiments, error bars represent s.e.m. (F) HEK293 cells and Hana3A were cotransfected with a GAL4-regulated luciferase reporter construct and chimeric constructs encoding the GAL4 DNA-binding domain alone (gal4), or fused to ß-arrestin2. Luciferase activity was measured 48 hours after transfection using equal amounts of total cellular lysates from untreated cells (ß-arr2) and from HEK293 (ß-arr2+B) and Hana3A (ß-arr2+B hana) cells that were treated with bourgeonal for 6 hours. Luciferase activity significantly increased upon hOR17-4 stimulation. For each panel, results of five independent experiments performed in triplicate were averaged and plotted, error bars represent s.e.m. Bars, 10 µm.

View larger version (49K): [in a new window]   Fig. 2. Redistribution of ß-arrestin2 in human spermatozoa upon bourgeonal application. Subcellular localization of ß-arrestin2 is changed upon bourgeonal stimulation of human spermatozoa. (A) Immunostaining of human sperm using anti-ß-arrestin2 antibodies (green). Before treatment (0) ß-arrestin2 is localized to the midpiece, with some minor staining in the head. After bourgeonal stimulation (20 min), ß-arrestin2 is translocated to the head, as indicated by increasing colocalization with DAPI (blue). Pretreatment with the hOR17-4 antagonist undecanal (+undecanal) blocks ß-arrestin2 redistribution in response to bourgeonal stimulation. Bar, 10 µm. (B) Specificity of the ß-arrestin2 antibody was confirmed by pre-incubation with a specific blocking peptide. Midpiece staining is completely abolished, and barely detectable staining in the head is observed (shown as overly with DAPI and without DAPI staining). The staining was similar to the background, which is present when only secondary antibodies are applied (2-ry antibody control). (C) Western blot analysis of fractionated human sperm showing that ß-arrestin2 is present in the nucleus after bourgeonal stimulation. Nonstimulated (-) and bourgeonal stimulated (+) human sperm were fractionated as described in the Materials and Methods, the purity of nuclear fraction (nuc) was verified with anti-ß-actin antibodies. Specificity of the ß-arrestin2 antibody was confirmed by preincubation with a blocking peptide, no ß-arrestin2 specific band (44 kDa) was detected under this condition. cyt, cytoplasmic fraction; mem, membrane fraction. (D) ß-arrestin2 signals in the different sperm fractions were quantified by densitometry. Averages from three independent experiments are shown, error bars represent s.e.m. (E) The ß-arrestin2 antibody detects endogenous ß-arrestin2 in HEK293 cells (contr), in human sperm lysate (sperm) and recombinant ß-arrestin2 in HEK293 cells transfected with ß-arrestin2 (ß-arr2). Anti-actin antibodies were used as a loading control. Preincubation with a blocking peptide confirms the specificity of the antibody labeling.

View larger version (44K): [in a new window]   Fig. 3. Odorant receptor stimulation results in phosphorylation and nuclear translocation of MAPK in human spermatozoa. (A) p42/44 and p38 MAPK are phosphorylated in response to bourgeonal stimulation of human sperm. Western blot analysis of total cell lysate of untreated human sperm cells (0) and cells that were treated with bourgeonal for 5 and 30 minutes. Lysates were probed for the presence of phosphorylated p44/42 MAPK and phosphorylated p38 MAPK using antibodies that specifically recognize the phosphorylated form of the proteins. Detection of the total amounts of p42/44 and p38 are shown as control. (B,C) p42/44 MAPK and p38 MAPK signals and signal from the unphosphorylated forms were quantified by densitometry. The amount of each phosphorylated MAPK was normalized to the total MAPK level in three independent experiments. Error bars represent s.e.m. (D) Phosphorylated p42/44 MAPK is present in the nuclei of bourgeonal-treated human sperm; detection of the total amount of p42/44 in the nuclear fraction is shown as a control. (E) The signal from phosphorylated p42/44 MAPK in the nuclear fraction was quantified by densitometry and normalized to a total amount p42/44 MAPK levels in two independent experiments. Error bars represent s.e.m.

View larger version (17K): [in a new window]   Fig. 4. Redistribution of hOR17-4 in human spermatozoa upon bourgeonal application. (A) Subcellular localization of hOR17-4 is changed upon bourgeonal stimulation of human spermatozoa. An antibody against hOR17-4 raised in mouse labeled the midpiece of non-treated human spermatozoa (0), some cells show a ring-like staining around the head; after bourgeonal stimulation (20 min) staining is strongly decreased in the midpiece. Treatment with undecanal blocks hOR17-4 redistribution, similar to ß-arrestin2. Bar, 10 µm. (B) Western blot analysis of human sperm with the anti-hOR17-4 antibody shows that the hOR17-4 protein can be detected at 40 kDa in the total cell lysate, but does not translocate to the nucleus in response to bourgeonal stimulation. (C) Control western blots showing the specificity of the antibody. Equal amounts of hOR17-4-GFP-transfected HEK293 cells and mock-transfected cells were probed with anti-hOR17-4 and anti-GFP antibody.

View larger version (33K): [in a new window]   Fig. 5. PKA is involved in hOR17-4 desensitization. (A) Immunostaining of human sperm using anti-PKA antibodies (green) shows, that PKA is localized to the midpiece and the tail in varying amounts, some cells also show ring-like staining around the head; nuclei are counterstained with DAPI. Bar, 10 µm. (B) Western blot analysis of human sperm lysate with anti-PKA phospho-substrate antibodies. Whereas non-stimulated cells (0) showed no staining with this antibody, cells that were treated with bourgeonal for 30 minutes showed labeling of a strong band of 40 kDa. (C) Representative ratiofluorometric recordings of the head and midpiece of individual human spermatozoa showing that PKA might be involved in hOR17-4 desensitization. The cytosolic Ca2+ level of Fura-2-loaded cells is depicted as the integrated fluorescence ratio (f340/f380) and viewed as a function of time. Cells were pre-incubated with PKA inhibitor H-89 in varying concentrations, bourgeonal was applied for 5 seconds in each case.

View larger version (21K): [in a new window]   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.