A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation

Abstract

The actin binding protein filamin A (FLNA) is required for somatostatin receptor 2 (SSTR2) and dopamine receptor 2 (DRD2) expression and signaling in GH- and PRL-secreting PitNETs, respectively, playing a role in tumor responsiveness to somatostatin receptors ligands and dopaminergic drugs. FLNA functions are regulated by several mechanisms, including phosphorylation. It has been shown that in GH-secreting PitNETs FLNA phosphorylation on Ser2152 (P-FLNA) switches FLNA function from a scaffold that allows SSTR2 signal transduction, to a signal termination protein that hampers SSTR2 antitumoral effects. Aims of the present study were to evaluate in PRL- and ACTH-secreting PitNETs cell lines MMQ and AtT-20 the effects of cAMP pathway activation and DRD2 agonist on P-FLNA and the impact of P-FLNA on DRD2 signal transduction. We found that forskolin increased (+2.2 ± 0.8-fold, p < 0.01 in MMQ; +1.9 ± 0.58-fold, p < 0.05 in AtT-20), and DRD2 agonist BIM53097 reduced (-49.4 ± 25%, p < 0.001 in MMQ; -45.8 ± 28%, p < 0.05 in AtT-20), P-FLNA on Ser2152. The overexpression of a phosphomimetic (S2152D) FLNA mutant in both cell lines prevented DRD2 antiproliferative effects, that were comparable in cells transfected with empty vector, wild-type FLNA as well as phosphodeficient FLNA mutant (S2152A) (-20.6 ± 5% cell proliferation, p < 0.001 in MMQ; -36.6 ± 12%, p < 0.01 in AtT-20). Accordingly, S2152D FLNA expression abolished the expected ability of BIM53097 to increase or decrease, in MMQ and in AtT20 respectively, ERK phosphorylation, an effect that was maintained in S2152A FLNA expressing cells (+1.8 ± 0.65-fold, p < 0.05 in MMQ; -55 ± 13%, p < 0.01 in AtT-20). In addition, the inhibitory effects of DRD2 on hormone secretion (-34.3 ± 6% PRL, p < 0.05 in MMQ; -42.8 ± 22% ACTH, p < 0.05 in AtT-20, in cells expressing S2152A FLNA) were completely lost in S2152D FLNA transfected cells. In conclusion, our data demonstrated that cAMP pathway and DRD2 agonist regulated FLNA activity by increasing or decreasing, respectively, its phosphorylation. Moreover, we found that P-FLNA prevented DRD2 signaling in PRL- and ACTH-secreting tumoral pituitary cell lines, suggesting that this FLNA modification might represent a new regulatory mechanism shared by different GPCRs. In PitNETs expressing DRD2, modulation of P-FLNA might suggest new pharmacological strategies to overcome drug resistance, and P-FLNA might represent a new biomarker for tumor responsiveness to dopaminergic agents.

Keywords: cAMP/PKA pathway; dopamine receptor type 2; filamin A phosphorylation; pituitary neuroendocrine tumors; signal transduction.

Figure 1

FLNA phosphorylation is reduced by DRD2 agonist and increased by forskolin treatment, respectively. MMQ cells (A, C) and AtT-20 cells (B, D) were treated with 1μM forskolin or 100 nM BIM53097 for 10 min (A, B) or indicated times (C, D) at 37°C. The graphs show the quantification of P-FLNA/total FLNA ratio. Experiments were repeated 5 times. Values represent mean ± S.D. normalized vs. respective basal. Representative immunoblots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 vs. corresponding basal.

Figure 2

FLNA S2152D mutant reverted DRD2 inhibitory effects on cell proliferation in both MMQ and AtT-20 cells. MMQ (A, C, E) and AtT-20 cells (B, D, F) were transiently transfected with empty vector, wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. MMQ (A) and AtT-20 (B) were than incubated 72 h or 96 h with 100 nM BIM53097, respectively. BrdU incorporation in newly synthesized DNA was measured. Experiments were repeated 4 times and each determination was done in quadruplicate. *p < 0.05, **p < 0.01, ***p < 0.001 vs. corresponding basal. (C–F) Cells were treated 3 h (C, D) or 48 h (E, F) with 100 nM BIM53097. The graphs show the quantification of cyclin D3 (C, D) or p27 (E, F) expression levels respectively, normalized to GAPDH. Representative immunoblots are shown. Data represent mean ± S.D. from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. corresponding basal.

Figure 3

Effects of S2152D FLNA mutant overexpression on ERK1/2 and AKT phosphorylation. MMQ (A, C) and AtT-20 cells (B) were treated 10 min with 100 nM BIM53097. The graphs show the quantification of P-ERK1/2 expression levels normalized to total ERK1/2 (A and B) and P-AKT/total AKT (C). Data represent mean ± S.D. from three independent experiments. Representative immunoblots are shown. *p < 0.05, **p < 0.01 vs. corresponding basal. ^§§p < 0.01 vs. corresponding BIM53097 treated.

Figure 4

FLNA phosphorylation abolished antisecretory effects of DRD2. MMQ (A) and AtT-20 cells (B) were transiently transfected with wild-type, S2152A and S2152D FLNA mutants for 72 h at 37°C. After 4 h (MMQ) or 24 h (AtT-20) BIM53097 treatment, hormonal assay was performed in order to detect hormones levels. The graphs show the percentage of PRL (A) and ACTH (B) secretion levels. Data represent mean ± S.D. from three independent experiments and each determination was done in triplicate. *p < 0.05, **p < 0.01 vs. corresponding basal.