On the selectivity of the Gαq inhibitor UBO-QIC: A comparison with the Gαi inhibitor pertussis toxin

Abstract

Gαq inhibitor UBO-QIC (FR900359) is becoming an important pharmacological tool, but its selectivity against other G proteins and their subunits, especially βγ, has not been well characterized. We examined UBO-QIC's effect on diverse signaling pathways mediated via various G protein-coupled receptors (GPCRs) and G protein subunits by comparison with known Gαi inhibitor pertussis toxin. As expected, UBO-QIC inhibited Gαq signaling in all assay systems examined. However, other non-Gαq-events, e.g. Gβγ-mediated intracellular calcium release and inositol phosphate production, following activation of Gi-coupled A1 adenosine and M2 muscarinic acetylcholine receptors, were also blocked by low concentrations of UBO-QIC, indicating that its effect is not limited to Gαq. Thus, UBO-QIC also inhibits Gβγ-mediated signaling similarly to pertussis toxin, although UBO-QIC does not affect Gαi-mediated inhibition or Gαs-mediated stimulation of adenylyl cyclase activity. However, the blockade by UBO-QIC of GPCR signaling, such as carbachol- or adenosine-mediated calcium or inositol phosphate increases, does not always indicate inhibition of Gαq-mediated events, as the βγ subunits released from Gi proteins following the activation of Gi-coupled receptors, e.g. M2 and A1Rs, may produce similar signaling events. Furthermore, UBO-QIC completely inhibited Akt signaling, but only partially blocked ERK1/2 activity stimulated by the Gq-coupled P2Y1R. Thus, we have revealed new aspects of the pharmacological interactions of UBO-QIC.

Keywords: FR900359; G protein; GPCR; Gα(q) inhibitor; Gβγ subunits; UBO-QIC.

Figure 1

Chemical structures of naturally-occuring cyclic depsipeptides UBO-QIC (FR900359) and YM-254890.

Figure 2

Selectivity of UBO-QIC for Gα_q- versus Gα_s- and Gα_i-mediated signaling. A. NECA-induced cAMP accumulation in CHO cells expressing the recombinant human A_2BAR (Gα_s). B. NECA-induced inhibition of forskolin-stimulated (10 μM) accumulation of cAMP in CHO cells expressing the recombinant human A₁AR (Gα_i). Cells were treated with agonist for 20 min and forskolin for 10 min. C. 2MeSADP-induced inhibition of forskolin-stimulated (10 μM) accumulation of cAMP in 1321N1 astrocytoma cells expressing the human P2Y₁₂ receptors. D. Carbachol-inducd intracellular calcium mobilization in CHO cells expressing the recombinant human M3 muscarinic receptor (Gα_q). For all assays, cells were pretreated with UBO-QIC (300 nM) for 30 min or PTX (200 ng/ml) overnight before the addition of agonists. Results are expressed as mean ± SEM and are from at least three independent experiments performed in duplicate or triplicate. The EC₅₀ values from agonist response curves are listed in the text. ^#Significantly different from control or lower concentrations of NECA in the presence of PTX (P<0.05, One-Way ANOVA with post-hoc test).

Figure 3

Intracellular calcium mobilization mediated via the G_i-coupled A₁AR (A) and M₂ muscarinic receptor (B), both expressed in CHO cells, and the G_q-coupled P2Y₁ (C) and G_i-coupled P2Y₁₂ nucleotide receptors (D) expressed in 1321N1 astrocytoma cells. Cells were pretreated with UBO-QIC (10 and 100 nM) for 30 min or PTX (200 ng/ml) overnight before the addition of agonists. Results were expressed as mean ± SEM from 3 independent experiments performed in duplicate.

Figure 4

Stimulation of IP1 production via activation of G_i-coupled A₁AR (A) and M₂ muscarinic receptor (B) expressed in CHO cells, or G_q-coupled M₃ muscarinic receptors in CHO cells (C) and G_q-coupled P2Y₁ nucleotide receptor in 1321N1 astrocytoma cells (D). Cells were pretreated with UBO-QIC (100 nM) for 30 min and PTX (200 ng/ml) overnight before the addition of agonists. Results were expressed as mean ± SEM from 3-4 separate experiments performed in duplicate.

Figure 5

Stimulation of ERK1/2 activity in CHO cells expressing the A₁AR (A), M₂ (B) or M₃ (D) muscarinic receptor, and in 1321N1 astrocytoma cells expressing the P2Y₁R (C). Cells were pretreated with UBO-QIC (100 nM) or GO6983 (10 μM) for 30 min or PTX (200 ng/ml) overnight before the addition of agonists (5 min incubation). Data are mean ± SEM from three experiments performed in duplicate. #Significantly different from the corresponding Control values in the absence of UBO (P<0.05).

Figure 6

Effect of UBO-QIC on the Akt1/2/3 phosphorylation stimulated by Gαq-coupled P2Y₁R (A, 1321N1 cells) or Gi-coupled A₁AR (B, CHO cells) or P2Y₁₂R (C, 1321N1 cells). A. Cells were pretreated with UBO-QIC (100 nM) for 30 min before the addition of agonists. B. Cells were pretreated with UBO-QIC (100 nM) for 30 min or PTX (200 ng/ml) overnight. C. Cells were pretreated with UBO-QIC (100 nM and 300 nM) for 30 min or PTX (200 ng/ml) overnight before the addition of agonist. Results are expressed as mean ± SEM from 3 independent experiments performed in duplicate. ^#Significantly different from corresponding control values (P<0.05).

Figure 6

Effect of UBO-QIC on the Akt1/2/3 phosphorylation stimulated by Gαq-coupled P2Y₁R (A, 1321N1 cells) or Gi-coupled A₁AR (B, CHO cells) or P2Y₁₂R (C, 1321N1 cells). A. Cells were pretreated with UBO-QIC (100 nM) for 30 min before the addition of agonists. B. Cells were pretreated with UBO-QIC (100 nM) for 30 min or PTX (200 ng/ml) overnight. C. Cells were pretreated with UBO-QIC (100 nM and 300 nM) for 30 min or PTX (200 ng/ml) overnight before the addition of agonist. Results are expressed as mean ± SEM from 3 independent experiments performed in duplicate. ^#Significantly different from corresponding control values (P<0.05).

Figure 7

MRS2365-induced intracellular calcium mobilization in HEK293 cells expressing an endogenous P2Y₁R (A) and CPA- induced intracellular calcium mobilization in DDT1-MF2 cells with an endogenous A₁AR (B). Cells were pretreated with UBO-QIC (100 nM) for 30 min or PTX (200 ng/ml) overnight before the addition of agonist. All data are in relative fluorescence units and are expressed as mean ± SEM from 3 experiments performed in triplicate.

Figure 8

GPCR signaling pathways and their interaction with inhibitors.