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. 2013 Sep 10:186:62-76.
doi: 10.1016/j.regpep.2013.06.009. Epub 2013 Jun 29.

Desensitization of human CRF2(a) receptor signaling governed by agonist potency and βarrestin2 recruitment

Richard L Hauger  1 , J Alberto Olivares-Reyes, Sandra Braun, Judith Hernandez-Aranda, Christine C Hudson, Eric Gutknecht, Frank M Dautzenberg, Robert H Oakley
Affiliations

Desensitization of human CRF2(a) receptor signaling governed by agonist potency and βarrestin2 recruitment

Richard L Hauger et al. Regul Pept. .

Abstract

The primary goal was to determine agonist-specific regulation of CRF2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF1 receptor signaling. In transfected HEK293 cells, activation of CRF2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100nM) produced strong βarrestin2 translocation and colocalization with membrane CRF2(a) receptors while CRF (1μM) generated only weak βarrestin2 recruitment. βarrestin2 did not internalize with the receptor, however, indicating that transient CRF2(a) receptor-arrestin complexes dissociate at or near the cell membrane. Since deletion of the βarrestin2 gene upregulated Gs-coupled CRF2(a) receptor signaling in MEF cells, a βarrestin2 mechanism restrains Gs-coupled CRF2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, βarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response.

Keywords: CK1; CK2; CRF receptor type 1; CRF receptor type 2(a); CRF(1) receptor; CRF(2(a)) receptor; Corticotropin releasing factor receptor (CRF); G protein-coupled receptor; GPCR; GPCR kinase; GRK; K44A; PKA; PKC; SCP; SRP; Urocortin 1 (UCN1); Urocortin 2 (UCN2); Urocortin 3 (UCN3); casein kinase 1; casein kinase 2; dynamin dominant negative mutant; protein kinase A; protein kinase C; stresscopin; stresscopin-related peptide; βarrestin2.

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Conflict of interest statement

Conflict of Interest

The authors have no conflict of interest to report.

Figures

Figure 1
Figure 1. Effect of homologous CRF2(a) receptor desensitization on sensitivity (EC50) and maximum for the concentration-response stimulation of intracellular cyclic AMP accumulation by urocortin 2
Homologous CRF2(a) receptor desensitization was induced by pretreating Y79 cells with UCN2 (100 nM) or UCN3 (100 nM) for 30 min. Cyclic AMP levels (pmoles/106 cells) were then measured in triplicate in desensitized and control cells stimulated with 0–100 nM UCN2 for 15 min. By ANOVA, there was a significant interaction [F=68.0; p<0.0001] for group (Control or Urocortin Pretreatment) and stimulation conditions (10−11 to 10−7 M UCN2). A significant within group effect [F=114.5; p<0.0001] was found with planned comparisons indicating the following: ap<0.001 vs Basal (no UCN2). A significant across pretreatment group effect [F=208.8; p<0.0001] was detected with planned comparisons indicating the following differences: bp<0.001 vs Control (no pretreatment); cp<0.05 vs UCN2 pretreatment. These findings were confirmed in four independent experiments.
Figure 2
Figure 2. Concentration-dependence of homologous CRF2 receptor desensitization induced by urocortin 2 or 3
After Y79 cells were pretreated with UCN2 (A), UCN3 (B), or buffer (Control), cyclic AMP levels (pmoles/106 cells) were measured in washed cells following an additional incubation with buffer (Basal) or maximal stimulation with 100 nM UCN2 for 15 min. Results were obtained in five independent experiments (n = 10 replicates per pretreatment group). By ANOVA, there was a significant interaction [F=77.41; p<0.0001] for pretreatment group (Urocortin concentration) and stimulation condition (Basal – no UCN2 or +100 nM UCN2). There were also significant within group differences for UCN2 Pretreatment [F=514.1; p<0.0001] and UCN3 Pretreatment [F=1492; p<0.0001]. Planned comparisons detected significant differences in UCN2-stimulated cyclic AMP stimulation within UCN2 Pretreatment groups (ap<0.001 vs Control; bp<0.01 vs 1 nM UCN2; cp<0.001 vs 1 nM UCN2) and within UCN3 Pretreatment groups (ap<0.001 vs Control; dp<0.01 vs Control; ep<0.01 vs 10 nM UCN3; fp<0.001 vs 10 nM UCN3; gp<0.05 vs 100 nM UCN3). When the calculated Δ cyclic AMP responses (+100 nM UCN2 stimulation - Basal cyclic AMP levels) for the UCN2 and UCN3 Pretreatment groups were compared, the ANOVA detected significant across group (UCN2 vs UCN3 Pretreatment) interactions [F=90.42; p<0.0001], with planned comparisons indicating significantly greater desensitization by UCN2 than UCN3 at the following concentrations: hp<0.001 vs 100 nM UCN3; ip<0.01 vs 1 µM UCN3; ; jp<0.01 vs 1 µM UCN3.
Figure 3
Figure 3. Effect of CRF1 and CRF2 receptor antagonists on UCN2-stimulated cyclic AMP accumulation
(A) The effects of 15-min pretreatment with the selective CRF2 receptor antagonist antisauvagine-30 (ASV-30 100 nM), UCN2 (100 nM), or buffer (Control) on UCN2-stimulated cyclic AMP accumulation were compared. Data (mean ± SEM) are cyclic AMP levels (pmoles/106 cells; n = 9) measured in cells incubated with buffer (Basal) or maximally stimulated with 100 nM UCN2 for 15 min (+100 nM UCN2) after the pretreatment period. By ANOVA, there was a significant interaction [F=94.43; p<0.0001] for pretreatment group (Control, UCN2, ASV-30) and stimulation conditions (Basal – no UCN2 or +100 nM UCN2). A significant within group effect [F=750.7; p<0.0001] was observed with planned comparisons indicating the following: ap<0.001 vs Basal. There was also a significant effect [F=63.88; p<0.0001] across the pretreatment groups with planned comparisons indicating the following differences in +100 nM UCN2 stimulation: bp<0.001 vs Control; cp<0.001 vs ASV-30. These results were replicated in two independent experiments. (B) After Y79 cells were pretreated with 100 nM UCN2 or buffer (Control) for 30 min in the presence or absence of the selective CRF1 receptor antagonist NBI-30775, cyclic AMP levels (pmoles/106 cells; n = 9) were measured in washed cells incubated with buffer (Basal) or maximally stimulated with 100 nM UCN2 for 15 min (+100 nM UCN2). By ANOVA, there was a significant interaction [F=265.4; p<0.0001] for pretreatment group (Control, UCN2, NBI-30775, NBI-30775 + UCN2) and stimulation condition (Basal or +100 nM UCN2). A significant within group effect was observed with planned comparisons indicating the following: ap<0.001 vs Basal. There was also a significant effect [F=239.2; p<0.0001] across the pretreatment groups with planned comparisons indicating the following differences in +100 nM UCN2 stimulation: bp<0.001 vs Control; cp<0.001 vs NBI-30775. These results were replicated in two independent experiments.
Figure 4
Figure 4. Comparison of the magnitude of homologous CRF2 receptor desensitization induced by urocortins and CRF
(A) After Y79 cells were pretreated with 100 nM UCN2, UCN3, r/hCRF, or oCRF for 30 min, cyclic AMP levels (pmoles/106 cells; n = 6) were measured in washed cells incubated with buffer (Basal) or maximally stimulated for 15 min (+100 nM UCN2). By ANOVA, there was a significant interaction [F=64.04; p<0.0001] for pretreatment group (Control, UCN2, UCN3, CRF) and stimulation condition (Basal or +100 nM UCN2). A significant within group effect was found with planned comparisons indicating the following: ap<0.001 vs Basal. There was also a significant effect [F=239.2; p<0.0001] across pretreatment groups with planned comparisons indicating the following differences for +100 nM UCN2 stimulation: bp<0.001 vs Control; cp<0.001 vs r/hCRF; dp<0.001 vs oCRF. These results were replicated in five independent experiments. (B) After Y79 cells were pretreated for 1 h with buffer (Control) or 100 nM urocortin 1 (UCN1) in the presence or absence of the selective CRF1 receptor antagonist NBI-30775, they were then incubated with buffer (Basal) or maximally stimulated with 100 nM UCN2 for 15 min and cyclic AMP levels (pmoles/106 cells, n=9) measured. By ANOVA, there was a significant interaction [F=197.1; p<0.0001] for pretreatment group (Control, UCN1, NBI-30775, NBI-30775 + UCN1) and stimulation condition (Basal or +100 nM UCN2). A significant within group effect was found with planned comparisons indicating the following: ap<0.001 vs Basal. There was also a significant effect [F=138.9; p<0.0001] across the pretreatment groups with planned comparisons indicating the following differences for +100 nM UCN2 stimulation: bp<0.001 vs Control; cp<0.001 vs NBI-30775. These results were replicated in three independent experiments.
Figure 5
Figure 5. Effect of homologous CRF2 receptor desensitization on the ability of forskolin, PACAP38, and isoproterenol to stimulate intracellular cyclic AMP accumulation
(A) After homologous CRF2 receptor desensitization was induced by exposing Y79 cells to UCN2, cyclic AMP levels (pmoles/106 cells) were then measured in desensitized and control (no UCN2 pretreatment) cells incubated for 15 min with buffer (Basal), UCN2, or forskolin (n=10/condition). By ANOVA, there was a significant interaction [F=5.88; p<0.005] for pretreatment group (Control, UCN2) and stimulation condition (Basal, +UCN2, or +Forskolin). A significant within group effect [F=709.7; p<0.0001] was found with planned comparisons indicating the following differences: ap<0.001 vs Basal; bp<0.001 vs Forskolin. A significant effect [F=3.59; p<0.05] was detected across the two pretreatment groups with planned comparisons indicating the following difference for +100 nM UCN2 stimulation: cp<0.001 vs Control. These findings were replicated in two independent experiments. (B) After Y79 cells were pretreated with buffer (Control) or 100 nM UCN2 for 30 min, cyclic AMP responses (%Control) to a 15-min stimulation with 100 nM UCN2, 100 nM PACAP38, or 10 µM isoproterenol (n = 12/group) were then measured in control and desensitized. By ANOVA, there were significant differences across the Control and UCN2 Pretreatment groups [F= 108.2, p < 0.0001] with planned comparisons indicating the following: ap<0.001 vs Control. These findings were replicated in two independent experiments.
Figure 6
Figure 6. Independence of homologous desensitization processes controlling signal transduction by CRF1 and CRF2 receptors co-expressed in Y79 cells
(A) The effect of prolonged CRF exposure on Gs-coupled CRF2 receptor signaling was determined by pretreating Y79 cells with buffer (Control) or 100 nM oCRF for 1 h (to induce maximal CRF1 receptor desensitization [20]). Cyclic AMP accumulation (pmoles/106 cells; n = 9) was then measured after CRF2(a) receptors (CRF-R2a) were maximally stimulated by UCN2 (+100 nM UCN2) or CRF1 receptors (CRF-R1) were maximally stimulated by CRF (+100 nM oCRF) for 15 min. By ANOVA, there was a significant interaction [F=27.92; p<0.0001] for pretreatment conditions (Control, CRF) and stimulation group (Basal, +100 nM UCN2 or +100 nM oCRF). A significant within group effect was found with planned comparisons indicating the following: ap<0.001 vs Basal. There was also a significant effect across the pretreatment groups with planned comparisons indicating the following differences for +100 nM CRF stimulation: bp<0.001 vs Control. These results were replicated in three independent experiments. (B) The effect of prolonged urocortin exposure on Gs-coupled CRF1 receptor signaling was assessed in Y79 cells by incubation with buffer (Control), or pretreatment with 100 nM UCN2 or UCN3 for 1 h (to induce maximal CRF2 receptor desensitization). Cyclic AMP accumulation (pmoles/106 cells; n = 13) was then measured after CRF1 receptors (CRF-R1) were maximally stimulated by CRF (+100 nM oCRF) or CRF2(a) receptors (CRF-R2a) were maximally stimulated by UCN2 (+100 nM UCN2) for 15 min. By ANOVA, there was a significant interaction [F=11.20; p<0.0001] for pretreatment conditions (Control, UCN2, UCN3) and stimulation group (Basal, +100 nM UCN2 or +100 nM oCRF). A significant within group effect was found with planned comparisons indicating the following: ap<0.001 vs Basal. There was also a significant effect across the pretreatment groups with planned comparisons indicating the following differences for +100 nM UCN2 stimulation: bp<0.001 vs Control. These results were replicated in three independent experiments. These results were replicated in four independent experiments.
Figure 7
Figure 7. Agonist-stimulated phosphorylation of membrane CRF2(a) receptors
(A) 72 h after transfection with HA-tagged CRF2(a) receptor cDNA, HEK293 cells were metabolically labeled with 32Pi and then stimulated with 100 nM UCN2 for 0–60 min. By ANOVA with planned comparisons, the levels of phosphorylated CRF2(a) receptor (Mr 60–70 kDa) measured by densitometry were significantly increased: ap<0.05 vs 0 min UCN2 (Control); bp<0.01 vs 0 min (Control). This data was replicated in three independent experiments. (B) The magnitude of CRF2(a) receptor phosphorylation was compared following a 10 min stimulation with buffer (Control) or 100 nM agonist (r/hCRF, UCN2, UCN3). ANOVA with planned comparisons detected the following significant differences in CRF2(a) phosphorylation bands: ap<0.01 vs Control; bp<0.001 vs Control; cp<0.01 vs r/hCRF. This data was replicated in three independent experiments.
Figure 8
Figure 8. The role of protein kinase A and casein kinases in the regulation of Gs-coupled CRF2(a) receptor signaling
(A) Effect of protein kinase A (PKA) inhibition on homologous CRF2(a) receptor desensitization. In the pretreatment period, Y79 cells were first incubated with media or H89 (2 µM) for 30 min and then exposed to media (Control) or UCN2 (100 nM) in the continuing presence of H89 for an additional 15 min. Pretreated cells were then incubated with buffer (Basal, n=3) or stimulated with UCN2 (+100 nM UCN2, n=10) for an additional 15 min. By ANOVA, there was a significant interaction [F=17.09; p<0.0001] for pretreatment group (Control, UCN2, H89, H89 + UCN2) and stimulation condition (Basal or +100 nM UCN2). A significant within group effect [F=77.27; p<0.0001] was found with planned comparisons indicating the following: ap<0.001 vs Basal. A significant effect [F=12.38; p<0.0001] was found across the pretreatment groups with planned comparisons indicating the following differences in +100 nM UCN2 stimulation: bp<0.01 vs. Control; cp<0.001 vs. H89. This data was replicated in three independent experiments. (B) Effect of PKA activators on UCN 2-stimulated cyclic AMP accumulation. After Y79 cells (n=13/group) were pretreated for 1 h with dibutyryl-cyclic AMP (dbcAMP) (4 mM) or the Sp-isomer of adenosine-3’,5’-cyclic AMP monophosphorothioate (Sp-cAMPS) (500 µM) to maximally activate PKA, they were incubated with buffer or stimulated with 100 nM UCN 2 for 15 min. By ANOVA, a significant within (but not across) pretreatment group effect [F=676.5; p<0.0001] was found with planned comparisons indicating the following: ap<0.001 vs. Control. These findings were replicated in two independent experiments. (C) Effect of casein kinase (CK) inhibition on UCN2-induced CRF2(a) receptor desensitization. Y79 cells were incubated for 30 min with media (Control) or CK inhibitors TBB (20 µM), Quinalizarin (20 µM) or IC261 (100 µM). After these cells were further pretreated with media or 100 nM UCN2 for 15 min (to desensitize CRF2(a) receptors) in the continuing presence of CK inhibitors, they were incubated with buffer or stimulated with 100 nM UCN2 for 15 min. By ANOVA, there was only a significant within group (Control and CK Inhibitor) difference [F= 62.98, p < 0.0001] with planned comparisons indicating the following: ap<0.001 vs. Control. Data (% control mean ± SEM) were replicated in two independent experiments (n = 6–10/group). (D) Role of PKA or casein kinase in CRF2(a) receptor phosphorylation. After cells were pretreated for 30 min with a PKA inhibitor (2 µM H89 or 100 µM RpcAMP) or a CK inhibitor (TBB 20 µM), they stimulated with 100 nM UCN2 for 10 min. Cell were also incubated with only dbcAMP (4 mM for 10 min) to activate PKA. CRF2(a) receptor phosphorylation was then quantitated (see Methods). This data was replicated in a separate experiment.
Figure 9
Figure 9. Agonist-stimulated CRF2(a) receptor internalization in HEK293 cells
(A) Flow cytometry was used to measure the time course for internalization of HA-tagged CRF2(a) receptors in HEK293 cells transfected 72 h earlier and then incubated with 100 nM UCN2 for 0-60 min (n=2–4/timepoint). By ANOVA, there were significant differences across the timepoint groups [F= 429.2; p < 0.0001] with planned comparisons indicating the following: ap<0.001 vs 0 min. This data was confirmed in five independent experiments. (B) CRF2(a) receptor internalization was measured in transfected HEK293 stimulated for 30 min with 100 nM UCN2, UCN3, r/hCRF, or oCRF (n=2–3/group). By ANOVA, there were significant differences across the agonist stimulation groups [F= 56.65; p < 0.0001] with planned indicating the following: ap<0.001 vs oCRF; bp<0.001 vs r/hCRF; cp<0.05 vs UCN3; dp<0.05 vs oCRF. This data was confirmed in three independent experiments. (C) After CRF2(a)-expressing HEK293 cells were transfected 72 h earlier with the dynamin dominant negative mutant K44A or empty vector, CRF2(a) receptor internalization was measured by flow cytometry following a 30-min stimulation with 100 nM UCN2 (n=2/group). By t-test, the groups were statistically different: ap<0.0001 vs Control. These results were replicated in four independent experiments. Vasopressin V2 receptor internalization in V2-expressing HEK293 cells incubated with 100 nM arginine vasopressin for 30 min was also significantly decreased (p=0.02) by dynamin K44A (29.8±3.7%) compared to control vector (50.0±0.2%).
Figure 10
Figure 10. Recruitment of βarrestin2 by agonist-activated human CRF2(a) receptors in HEK293 cells
Confocal microscopy was used to evaluate the interaction of βarrestin2-GFP with full-length wild-type CRF2(a) receptors transiently expressed in HEK293 cells. This representative experiment shows the distribution of βarrestin2-GFP in untreated cells and cells incubated with UCN2 (100 nM), UCN3 (100 nM), or r/hCRF (100 nM or 1 µM) for approximately 10 min.
Figure 11
Figure 11. Colocalization of βarrestin2 with cell-surface but not internalized agonist-activated CRF2(a) receptors
HEK293 cells transiently expressing βarrestin2-GFP and HA-CRF2(a) receptors were prelabeled with Alexa Fluor 594 conjugated anti-HA monoclonal antibody and evaluated by confocal microscopy. (A) Distribution of CRF2(a) receptor immunofluorescence (red) and βarrestin2-GFP fluorescence (green) in the same cells before and after a 2 min treatment with 100 nM UCN2 or UCN3. (B) Distribution of CRF2(a) receptor immunofluorescence (red) and βarrestin2-GFP fluorescence (green) after an ~30 min treatment with 100 nM UCN2 or UCN3. Colocalization of βarrestin2-GFP with the receptor is indicated by the yellow color in the overlay.
Figure 11
Figure 11. Colocalization of βarrestin2 with cell-surface but not internalized agonist-activated CRF2(a) receptors
HEK293 cells transiently expressing βarrestin2-GFP and HA-CRF2(a) receptors were prelabeled with Alexa Fluor 594 conjugated anti-HA monoclonal antibody and evaluated by confocal microscopy. (A) Distribution of CRF2(a) receptor immunofluorescence (red) and βarrestin2-GFP fluorescence (green) in the same cells before and after a 2 min treatment with 100 nM UCN2 or UCN3. (B) Distribution of CRF2(a) receptor immunofluorescence (red) and βarrestin2-GFP fluorescence (green) after an ~30 min treatment with 100 nM UCN2 or UCN3. Colocalization of βarrestin2-GFP with the receptor is indicated by the yellow color in the overlay.
Figure 12
Figure 12. Quantitation of βarrestin2 translocation to the agonist-activated CRF2(a) receptors in U2OS cells
U2OS cells transiently co-expressing βarrestin2-GFP and full-length wild-type human CRF2(a) receptors were plated in 96-well plates and cultured overnight. Cells were then stimulated with urocortin 2 or urocortin 3 (0–1000 nM) for 30 min and analyzed on the INCell Analyzer 3000. The reported parameter “Fgrains” represents the fluorescence intensity of βarrestin2-GFP localized with the receptor in spots or grains at the plasma membrane. Data represent the mean ± SEM for 3 independent experiments performed in triplicate. By ANOVA, there was a significant interaction [F=10.50; p<0.0001] for the urocortin group (UCN2, UCN3) and stimulation concentrations (10−11 to 10−6 M agonist). A significant within group effect [F=78.49; p<0.0001] was found with planned comparisons indicating significant increases in βArrrestin2 translocation over the agonist concentration range for both urocortins. A significant across pretreatment group effect [F=23.80; p<0.05] was detected with planned comparisons indicating the following: ap<0.001 vs UCN3.
Figure 13
Figure 13. Dose-response stimulation of cyclic AMP accumulation by urocortin 2 in wild-type and βarrestin2 knockout mouse embryonic fibroblast (MEF) cells
In two experiments, cyclic AMP levels (pmoles/well) were measured in quadruplicate in CRF2(a) receptor-expressing MEF cells stimulated with 0–100 nM UCN2 for 15 min. By ANOVA, there was a significant interaction [F=50.13; p<0.0001] for MEF cell group and stimulation conditions (10−12 to 10−7 M UCN2). A significant within group effect [F=251.9; p<0.0001] was observed with planned comparisons indicating significant increases in cyclic AMP accumulation over the UCN2 concentration range. A significant across MEF cell group effect [F=534.7; p<0.0001] was detected with planned comparisons indicating the following difference: ap<0.001 vs Wild-type MEF cells.
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