Differential effects of AGS3 expression on D(2L) dopamine receptor-mediated adenylyl cyclase signaling

Abstract

Activator of G protein signaling 3 (AGS3) binds Gα(i) subunits in the GDP-bound state, implicating AGS3 as an important regulator of Gα(i)-linked receptor (e.g., D2 dopamine and μ-opioid) signaling. We examined the ability of AGS3 to modulate recombinant adenylyl cyclase (AC) type 1 and 2 signaling in HEK293 cells following both acute and persistent activation of the D(2L) dopamine receptor (D(2L)DR). AGS3 expression modestly enhanced the potency of acute quinpirole-induced D(2L)DR modulation of AC1 or AC2 activity. AGS3 also promoted desensitization of D(2L)DR-mediated inhibition of AC1, whereas desensitization of D(2L)DR-mediated AC2 activation was significantly attenuated. Additionally, AGS3 reduced D(2L)DR-mediated sensitization of AC1 and AC2. These data suggest that AGS3 is involved in altering G protein signaling in a complex fashion that is effector-specific and dependent on the duration of receptor activation.

Fig. 1

Effect of AGS3 expression on acute regulation of AC isoforms. a Acute inhibition of A23187 (10 μM)-stimulated cAMP accumulation was measured following incubation with quinpirole as indicated in HEK293 cells stably expressing AC1 and the D_2LDR, or AC1 and the D_2LDR together with AGS3-Venus. b Cyclic AMP accumulation was quantified in HEK293 cells stably expressing AC2 and the D_2LDR with or without AGS3-Venus following acute treatment with PMA (1 μM) and increasing concentrations of quinpirole as indicated. Data points represent mean ± S.E.M. of at least three independent experiments performed in duplicate

Fig. 2

The effect of AGS3 expression on functional desensitization of D_2LDR-modulated AC1 activity. HEK293 cells stably expressing AC1, the D_2LDR, and AGS3-Venus were treated with quinpirole (1 μM) or vehicle for 2 h, followed by selective AC1 stimulation with A23187 (10 μM) and activation of the D_2LDR with quinpirole (1 μM). Data represent the mean ± SEM of three independent experiments

Fig. 3

The effect of AGS3 expression on functional desensitization of D_2LDR-modulated AC2 activity. HEK293 cells stably expressing AC2 and the D_2LDR alone, or together with AGS3-Venus or AGS3-Q/A-Venus were treated with quinpirole (1 μM) or vehicle for 2 h, followed by stimulation with PMA (1 μM) and quinpirole (1 μM). Data represent the mean ± SEM of at least three independent experiments. **p < 0.01, compared with AC2 + D_2L condition, one way analysis of variance followed by Dunnett’s post hoc test

Fig. 4

Effect of AGS3 expression on heterologous sensitization of AC isoforms. a HEK293 cells stably expressing AC1 and D_2LDR were stimulated with vehicle or quinpirole (1 μM) for 2 h, washed, and subsequently stimulated with A23187 (10 μM) as indicated. Data represent mean ± SEM of three independent experiments. *p < 0.05, unpaired t test. b HEK293 cells stably expressing AC2 and D_2LDR in the absence of, or coexpressed with AGS3-Venus or AGS3-Q/A-Venus as indicated were pretreated with vehicle or quinpirole (1 μM) for 2 h, washed, and subsequently stimulated with PMA (1 μM). Data represent mean ± S.E.M. of at least three independent experiments. **p < 0.01, one way analysis of variance followed by Dunnett’s post hoc test