Genome-Wide Small Interfering RNA Screening Reveals a Role for Cullin3-Really Interesting New Gene Ligase Signaling in Heterologous Sensitization of Adenylyl Cyclase

Abstract

Heterologous sensitization of adenylyl cyclase (AC) is revealed as enhanced or exaggerated AC/cAMP signaling that occurs following persistent activation of Gα _i/o-coupled receptors. This paradoxical phenomenon was discovered more than 40 years ago and was proposed as a cellular mechanism to explain the adaptive changes that occur following chronic exposure to drugs of abuse. However, the underlying molecular mechanisms of heterologous sensitization of AC remain largely unknown. In the present study, we performed a genome-wide cell-based RNA interference screen as an unbiased approach to identify genes associated with heterologous sensitization of AC. Following a series of validation and confirmation assays, three genes that form an E3 ligase complex, cullin3 (CUL3), neural precursor-cell-expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene (RING)-box protein 1 (RBX1), were identified as specific modulators of heterologous sensitization of AC. Furthermore, based on the downstream actions of these genes, we evaluated the activity of proteasome inhibitors as well as the specific NEDD8-activating enzyme inhibitor, MLN4924 (Pevonedistat), in AC sensitization. We demonstrate that MG-132 and bortezomib treatments could mimic the inhibitory effects observed with gene knockdown, and MLN4924 was potent and efficacious in blocking the development of heterologous sensitization of endogenous and recombinant AC isoforms, including AC1, AC2, AC5, and AC6. Together, by using genetic and pharmacological approaches, we identified, for the first time, cullin3-RING ligases and the protein degradation pathway as essential modulators for heterologous sensitization of AC. SIGNIFICANCE STATEMENT: Through a genome-wide cell-based RNA interference screening, we identified three genes that form an E3 ligase complex, cullin3, neural precursor-cell-expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene-box protein 1, as specific modulators of heterologous sensitization of AC. The effect of cullin3, NEDD8, or really interesting new gene-box protein 1 small interfering RNAs on heterologous sensitization was recapitulated by proteasome inhibitors, MG132 and bortezomib, and the specific NEDD8-activating enzyme inhibitor, MLN4924. These results suggest a novel hypothesis in which protein degradation is involved in the sensitization of AC signaling that occurs following chronic activation of Gα_i/o-coupled receptors.

Fig. 1.

Workflow of the development, execution, and validation of the cell-based genome-wide RNAi screen. (A) HEK-AC6/D2 cells were pretreated with vehicle or 1 μM quinpirole for 2 hours before being incubated with increasing concentrations of forskolin in the presence of spiperone (D2R antagonist) and IBMX (phosphodiesterase inhibitor) for 1 hour. (B) HEK-AC6/D2 cells were transfected for 70 hours with scrambled (Scr) or siRNAs targeting GNAS gene before being pretreated for 2 hours with vehicle or 1 μM quinpirole. After pretreatment, the cells were incubated with 300 nM forskolin in the presence of spiperone and IBMX. (C) Whole-cell lysates were prepared from HEK-AC6/D2 cells after transfection of scrambled (Scr) or GNAS-targeting siRNAs and probed with anti-Gα_s antibody. Immunoblot is representative of three independent experiments. (D) Workflow diagram of human genomewide RNAi screen and the subsequent validation assays.

Fig. 2.

Effect of siRNA for CUL3, NEDD8, or RBX1 genes on heterologous sensitization of AC and their protein expression. (A) Diagram of the cullin3-RING E3 ligase complex. S, substrate; BTB, BR-C, TTK, and BAB domain; Ub, ubiquitin. (B) HEK-AC6/D2 cells were transfected with scrambled (Scr) or pooled siRNAs targeting the CUL3, NEDD8, or RBX1 genes for 72 hours. Cells were pretreated for 2 hours with vehicle or 1 μM quinpirole. After pretreatment, the cells were incubated with 50 nM forskolin in the presence of 1 μM spiperone and 500 μM IBMX for 1 hour. Whole-cell lysates were prepared from HEK-AC6/D2 cells after transfection of scrambled (Scr) or indicated siRNAs and probed with anti-cullin3 (C), anti-NEDD8 (C), or anti-RBX1 (D) antibodies. Immunoblots are representative of three independent experiments. *P < 0.0001. Two-way ANOVA followed by Tukey’s test.

Fig. 3.

Analysis of gene-dose effects of cullin3 on heterologous sensitization of AC. HEKAC6/D2 cells were transfected with scrambled (Scr) or control vector (pcDNA 3.1+) and increasing quantities of CUL3 siRNA (A), Flag-DN-CUL3 DNA (B), or Myc-CUL3 DNA (C and D) as indicated. At 72 hours post–siRNA transfection or 48 hours post–DNA transfection, cells were pretreated for 2 hours (A–C) or indicated time (D) with vehicle or 1 μM quinpirole before incubating with 50 nM forskolin in the presence of 1 μM spiperone and 500 μM IBMX for 1 hour. Whole-cell lysates were prepared from HEK-AC6/D2 cells following the transfection conditions noted in panel above and probed with anti-cullin3 (A), anti-flag (B), or anti-myc (C) antibodies. Immunoblots are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA followed by Dunnett’s test (A). Two-way ANOVA followed by Tukey’s test (B and C). (D) *P < 0.05 vs. pcDNA 3.1+/Quin. Two-way ANOVA followed by Tukey’s test.

Fig. 4.

Effect of siRNA for CUL3, NEDD8, or RBX1 genes and MG-132 and bortezomib on heterologous sensitization of endogenous AC isoforms and AC6. (A) HEK-D2L cells were transfected with scrambled (Scr) or pooled siRNAs targeting the CUL3, NEDD8, or RBX1 genes for 72 hours. Cells were pretreated for 2 hours with vehicle or 1 μM quinpirole. After pretreatment, the cells were incubated with 1 μM forskolin in the presence of 1 μM spiperone and 500 μM IBMX for 1 hour. HEK-D2L (B) and HEK-AC6/D2 (C) cells were pretreated for 30 minutes with control (DMSO), 1 μM MG132, or 1 μM bortezomib before being treated with vehicle or 1 μM quinpirole for 2 hours. After treatment, HEK-D2L and HEK-AC6/D2 cells were incubated with 1 μM and 50 nM forskolin, respectively, in the presence of 1 μM spiperone and 500 μM IBMX for 1 hour. *P < 0.05; **P < 0.01; ***P < 0.001. Two-way ANOVA followed by Tukey’s test.

Fig. 5.

Effect and potency of MLN4924 on heterologous sensitization of endogenous AC isoforms and AC6. (A) Diagram for MLN4924 inhibition of neddylation of the cullin3-RING ligase. (B) HEK-D2L cells were pretreated for 30 minutes with control (DMSO) or 1 μM MLN4924. After pretreatment, the cells were incubated for 2 hours with vehicle or 1 μM quinpirole before being incubated with 1 μM forskolin in the presence of 1 μM spiperone and 500 μM IBMX for 1 hour. (C) HEK-AC6/D2 cells were pretreated for 30 minutes with control (DMSO) or 1 μM MLN4924 (left panel) or with control (DMSO) or increasing concentrations of MLN4924 (right panel) before being treated with vehicle or 1 μM quinpirole for 2 hours. After treatment, the cells were incubated with increasing concentrations of forskolin (left panel) or 50 nM forskolin (right panel) in the presence of 1 μM spiperone and 500 μM IBMX for 1 hour. (D) HEK-AC6/D2 cells were pretreated for 30 minutes with control (DMSO) or 1 μM MLN4924 before being treated for 2 hours with vehicle or 1 μM quinpirole. Whole-cell lysates were prepared and probed with anti-Cullin3 antibody. *P < 0.001. Two-way ANOVA followed by Tukey’s test.

Fig. 6.

Effect of MLN4924 on heterologous sensitization of recombinant and endogenous AC isoforms. (A) HEK-AC1/D2 or HEK-AC2/D2, (B) HEK-AC5/MOR1, or (C) NG108-15 cells were pretreated for 30 minutes with control (DMSO) or 1 μM MLN4924 as indicated. (A) Cells were then treated for 2 hours with vehicle or 1 μM quinpirole before being incubated with 1 μM A23187 (for AC1) or 500 nM phorbol 12-myristate 13-acetate (for AC2), respectively, in the presence of 1 μM spiperone and 500 μM IBMX for 1 hour. (B) HEK-AC5/MOR cells were treated for 2 hours with vehicle or 1 μM DAMGO (MOR agonist) before being incubated with 1 μM forskolin in the presence of 1 μM naltrexone (MOR antagonist) and 500 μM IBMX for 1 hour. (C) NG108-15 cells were treated for 18 hours with vehicle or 1 μM [D-Pen², D-Pen⁵] enkephalin (DOR agonist) before being incubated with 1 μM forskolin in the presence of 1 μM naltrindole (DOR antagonist) and 500 μM IBMX for 1 hour. *P < 0.05; ***P < 0.001. Two-way ANOVA followed by Tukey’s test.