Robust GRK2/3/6-dependent desensitization of oxytocin receptor in neurons

Abstract

Oxytocin plays critical roles in the brain as a neuromodulator, regulating social and other affective behavior. However, the regulatory mechanisms controlling oxytocin receptor (OXTR) signaling in neurons remain unexplored. In this study, we have identified robust and rapid-onset desensitization of OXTR response in multiple regions of the mouse brain. Both cell autonomous spiking response and presynaptic activation undergo similar agonist-induced desensitization. G-protein-coupled receptor kinases (GRK) GRK2, GRK3, and GRK6 are recruited to the activated OXTR in neurons, followed by recruitment of β-arrestin-1 and -2. Neuronal OXTR desensitization was impaired by suppression of GRK2/3/6 kinase activity but remained unaltered with double knockout of β-arrestin-1 and -2. Additionally, we observed robust agonist-induced internalization of neuronal OXTR and its Rab5-dependent recruitment to early endosomes, which was impaired by GRK2/3/6 inhibition. This work defines distinctive aspects of the mechanisms governing OXTR desensitization and internalization in neurons compared to prior studies in heterologous cells.

Keywords: biological sciences; molecular neuroscience; neuroscience.

Graphical abstract

Figure 1

Robust agonist-induced desensitization of OXTR response in the mouse brain (A) Cell-attached recordings from OXTR neurons in the LS (schematic on left) showing spikes in response to two sequential applications of 200 nM TGOT for 8 min each separated by 15 min. Sample traces (top) are shown for recordings during baseline (black), first application (blue), and second application (magenta). Summary graph of the time course (bottom left) and bar graphs with spike frequency at baseline, peak of first and peak of second response (bottom right) are also shown in this and subsequent panels. Spike frequency (Hz): baseline, 0.307 ± 0.130; 1^st response, 3.738 ± 0.594; 2^nd response, 0.281 ± 0.202; n = 8 cells, 4 mice. (B) Cell-attached recordings from OXTR neurons in the LS showing spikes in response to two sequential applications of 200 nM TGOT for 1 min separated by 15 min. Spike frequency (Hz): baseline, 0.697 ± 0.158; 1^st response, 7.000 ± 1.142; 2^nd response, 1.506 ± 0.273; n = 6 cells, 3 mice. (C) Whole-cell recordings of sIPSC from dorsal LS neurons showing response to two applications of 200 nM TGOT for 8 min each. sIPSC frequency (Hz): baseline, 3.350 ± 0.883; 1^st response, 12.890 ± 1.410; 2^nd response, 3.187 ± 1.006; n = 8 cells, 4 mice. (D) Whole-cell recordings of sIPSC from dorsal LS neurons showing response to two applications of 200 nM TGOT for 1 min separated by 15 min (n = 12 cells). sIPSC frequency (Hz): baseline, 2.849 ± 0.210; 1^st response, 13.250 ± 1.360; 2^nd response, 4.608 ± 0.814; n = 12 cells, 6 mice. (E) Cell-attached recordings from OXTR neurons in the PFC showing spikes in response to two sequential applications of 200 nM TGOT for 8 min each separated by 15 min. Spike frequency (Hz): baseline, 0.898 ± 0.377; 1^st response, 4.389 ± 0.917; 2^nd response, 0.561 ± 0.403; n = 6 cells, 4 mice. (F) Cell-attached recordings from OXTR neurons in the BNST showing spikes in response to two sequential applications of 200 nM TGOT for 1 min each separated by 15 min. Spike frequency (Hz): baseline, 0.369 ± 0.158; 1^st response, 3.962 ± 0.404; 2^nd response, 0.486 ± 0.128; n = 7 cells, 4 mice. The data in graphs are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, repeated measures one-way ANOVA with post-hoc Tukey’s multiple comparisons test.

Figure 2

OXTR-induced spiking and presynaptic release responses undergo desensitization (A) Cell-attached recordings from OXTR neurons in the LS in the presence of NBQX and picrotoxin (schematic on left) showing cell-autonomous spikes in response to two sequential applications of 200 nM TGOT for 8 min each separated by 15 min. Sample traces (top middle), summary graph of time course (bottom middle), and bar graphs with baseline, first peak response, and second peak response (right) are shown in this and subsequent panels. Spike frequency (Hz): baseline, 0.617 ± 0.284; 1^st response, 6.404 ± 1.093; 2^nd response, 0.344 ± 0.186; n = 9 cells, 5 mice. (B) Whole-cell recordings of sIPSC from dorsal LS neurons in the presence of NBQX in response to two sequential applications of 200 nM TGOT for 8 min each separated by 15 min. The responses indicate cell-autonomous activation of OXTR neurons in the LS. sIPSC frequency (Hz): baseline, 2.047 ± 0.336; 1^st response, 11.210 ± 1.693; 2^nd response, 1.861 ± 0.384; n = 6 cells, 3 mice. (C) Whole-cell recordings of mEPSC from OXTR neurons in the LS in response to two sequential applications of 200 nM TGOT for 8 min each separated by 15 min. The responses indicate presynaptic activation of OXTR on excitatory inputs to LS OXTR neurons. mEPSC frequency (Hz): baseline, 0.925 ± 0.136; 1^st response, 12.770 ± 2.286; 2^nd response, 1.656 ± 1.290; n = 7 cells, 6 mice. The data in graphs are shown as mean ± SEM. ∗∗p < 0.01, ∗∗∗∗p < 0.0001, repeated measures one-way ANOVA with post-hoc Tukey’s multiple comparisons test.

Figure 3

Desensitization of PI(4,5)P₂ hydrolysis and G protein activation, and recruitment of β-arrestin-1/2 and GRK2/3/6 to activated OXTR in primary neuronal cultures (A) Summary graph (bottom) of normalized BRET showing the time course of PI(4,5)P₂ hydrolysis downstream of OXTR activation by 200 nM TGOT (time of application is indicated by an arrow). Schematic (top) shows the BRET configuration with nanoLuc-PH-PLCδ and HaloTag-CAAX. Normalized BRET is reduced rapidly with TGOT application in control (black) followed by quick recovery suggesting desensitization (n = 3). Gα_q inhibitor UBO-QIC (1 μM, blue) abolished the response, whereas Gβγ inhibitor Gallein (40 μM, magenta) had no effect (n = 3–5 per group). (B) Summary graph (bottom) of ΔBRET showing the time course of G protein activation downstream of OXTR activation by 200 nM TGOT. Schematic (top) shows the BRET configuration with neurons expressing nanoLuc-Gγ, Gβ, and Gα_q along with GRK2-HaloTag. BRET is rapidly increased with TGOT application followed by quick recovery suggesting desensitization at the G protein level (n = 3). (C) Summary graph (bottom) of ΔBRET showing the time course of recruitment of β-arrestin-1 and β-arrestin-2 to neuronal OXTR activated by 200 nM TGOT. Schematic (top) shows the BRET configuration with neurons expressing OXTR-nanoLuc and β-arrestin-1 (blue) or β-arrestin-2 (magenta). BRET is increased with TGOT application suggesting recruitment of both β-arrestins to neuronal OXTR (n = 3–5 per group). (D) Summary graph (bottom) of ΔBRET time course shows that GRK2, GRK3, and GRK6 (but not GRK5) are recruited to neuronal OXTR activated by 200 nM TGOT. Schematic (top) shows the BRET configuration with neurons expressing OXTR-nanoLuc and GRK2-HaloTag (red), GRK3-HaloTag (blue), GRK5-HaloTag (gray), or GRK6-HaloTag (magenta) (n = 4 per group). The data in graphs are shown as mean ± SEM.

Figure 4

GRK2/3/6 are required while β-arrestins are redundant for neuronal OXTR desensitization (A) Whole-cell recordings of sIPSC from dorsal LS neurons of β-arrestin-1 KO mice showing response to two applications of 200 nM TGOT for 1 min each separated by 15 min. Sample traces for baseline (black), first response (blue), and second response (magenta) (top) and summary graph of time course (bottom) are shown. The lack of robust second response compared to first indicates intact OXTR desensitization. (B) Whole-cell recordings of sIPSC from dorsal LS neurons of β-arrestin-2 KO mice showing response to two applications of 200 nM TGOT for 1 min each separated by 15 min. Sample traces (top) and summary graph of time course (bottom) are shown. The lack of robust second response compared to first indicates intact OXTR desensitization. (C) Schematic showing the strategy for CRISPR/Cas9-based KO of β-arrestin-1 in the LS of constitutive β-arrestin-2 KO mice. Inset shows expression of GFP from AAV GFP-Cre and AAV gRNA-Syn-GFP in the LS. Scale bar: 400 μm. (D) Immunohistochemical confirmation of CRISPR/Cas9-induced depletion of β-arrestin-1 in the LS. Sections were stained with antibody against GFP or β-arrestin-1 in control and injected animals. Scale bar: 100 μm. (E) Immunoblots on LS lysates from WT, β-arrestin-1 KO (Arrb1^−/−), β-arrestin-2 KO (Arrb2^−/−), and CRISPR/Cas9-induced β-arrestin-1 KO (gArrb1) in β-arrestin-2 KO mice. Blots were stained with antibodies against β-arrestin-1, β-arrestin-2, and β-actin. (F) Whole-cell recordings of sIPSC from dorsal LS neurons of double β-arrestin KO mice showing response to two applications of 200 nM TGOT for 1 min each separated by 15 min. Sample traces (top) and summary graph of time course (bottom) are shown. The lack of robust second response compared to first indicates intact OXTR desensitization. (G) Whole-cell recordings of sIPSC from dorsal LS neurons in control condition (brown) and in the presence of CMPD101 (50 μM) and GRK6-IN-2 (50 μM) (blue). The summary graph of the time course of response to two applications of 200 nM TGOT for 1 min each separated by 15 min is shown (bottom). There is strong response to the second TGOT application in the presence of CMPD101 and GRK6-IN-2, indicating suppression of OXTR desensitization. Sample traces for CMPD101+GRK6-IN-2 condition are shown above the graph. (H) Bar graphs with baseline, first peak response, and second peak response in WT, β-arrestin-1 KO (Arrb1^−/−), β-arrestin-2 KO (Arrb2^−/−), double β-arrestin KO (Arrb2^−/−+ gArrb1), and GRK2/3/6 inhibition (CMPD101 + GRK6-IN-2). sIPSC frequency (Hz) WT: baseline, 2.920 ± 0.413; 1^st response, 13.100 ± 1.538; 2^nd response, 3.095 ± 0.314; n = 7 cells, 5 mice. sIPSC frequency (Hz) Arrb1^−/−: baseline, 4.136 ± 0.917; 1^st response, 12.590 ± 2.155; 2^nd response, 4.510 ± 1.003; n = 7 cells, 4 mice. sIPSC frequency (Hz) Arrb2^−/−: baseline, 2.813 ± 0.404; 1^st response, 13.150 ± 1.410; 2^nd response, 3.020 ± 0.277; n = 6 cells, 3 mice. sIPSC frequency (Hz) Arrb2^−/−+ gArrb1: baseline, 3.055 ± 0.486; 1^st response, 8.880 ± 1.342; 2^nd response, 3.927 ± 0.468; n = 10 cells, 6 mice. sIPSC frequency (Hz) CMPD101 + GRK6-IN-2: baseline, 2.600 ± 0.491; 1^st response, 15.410 ± 0.946; 2^nd response, 10.680 ± 1.506; n = 7 cells, 5 mice. The data in graphs are shown as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, Repeated measures two-way ANOVA with post-hoc Tukey’s multiple comparisons test.

Figure 5

Molecular mechanisms underlying agonist-induced internalization and trafficking of neuronal OXTR (A) Schematic depicting BRET assays for tracking OXTR internalization and trafficking in primary neuronal cultures. BRET was configured with OXTR-nanoLuc along with HaloTag-CAAX (for cell membrane localization), HaloTag-2xFYVE (early endosome), HaloTag-Rab11 (recycling endosome), HaloTag-Rab5 (Rab5 compartment), and HaloTag-Rab21 (Rab21 compartment). (B) Summary graph of normalized BRET showing the time course of neuronal OXTR internalization (HaloTag-CAAX), trafficking to early endosomes (HaloTag-2xFYVE) and moving into recycling endosomes (HaloTag-Rab11) following application of 200 nM TGOT (time of application is indicated by an arrow) (n = 3–9 per group). (C) Summary graph of normalized BRET showing the time course of neuronal OXTR trafficking to Rab5-but not Rab21-containing endosomes following application of 200 nM TGOT (n = 3–5 per group). (D) Trafficking of neuronal OXTR to early endosomes in response to 200 nM TGOT was impaired with expression of dominant negative Rab5 (Rab5 DN). Summary graph of the time course (left) of normalized BRET with OXTR-nanoLuc and HaloTag-2xFYVE. The bar graph of the averaged normalized BRET response for the last 5 min is shown on right. Control, 2.052 ± 0.128; Rab5 DN, 1.043 ± 0.041; n = 6–8 per group. (E) Internalization of neuronal OXTR in response to 200 nM TGOT was not affected with expression of dominant negative Rab5 (Rab5 DN). Summary graph of the time course (left) of normalized BRET with OXTR-nanoLuc and HaloTag-CAAX is shown. The bar graph of the averaged normalized BRET response for the last 5 min is shown on right. Control, 0.383 ± 0.023; Rab5 DN, 0.386 ± 0.017; n = 5 per group. (F) Summary graph (left) of normalized BRET showing the time course of neuronal OXTR internalization in the presence of vehicle (DMSO), Barbadin, CMPD101, GRK6-IN-1, GRK6-IN-2, and a mix of CMPD101 and GRK6-IN-1 (CMPD101+GRK6-IN-1) or a mix of CMPD101 and GRK6-IN-2 (CMPD101+GRK6-IN-2). BRET was configured with OXTR-nanoLuc and HaloTag-CAAX. The internalization was impaired in the presence of a mix of CMPD101 and GRK6-IN-2 but not with Barbadin, CMPD101, or GRK6-IN-2 alone. The bar graph of the averaged normalized BRET response for the last 5 min is shown on right. DMSO, 0.348 ± 0.027; CMPD101, 0.409 ± 0.046; GRK6-IN-1, 0.396 ± 0.029; GRK6-IN-2, 0.445 ± 0.017; CMPD101 + GRK6-IN-1, 0.703 ± 0.019; CMPD101 +GRK6-IN-2, 0.711 ± 0.027; Barbadin, 0.328 ± 0.022; n = 3–5 per group. The data in graphs are shown as mean ± SEM. ∗∗∗∗p < 0.0001, unpaired Student’s t test (D and E) or one-way ANOVA with post-hoc Dunnett’s test (F).