AC9 is less sensitive to Gαs compared with AC6 and is insensitive to Gβγ. (A) Dose-response curves of Gαs stimulation of membranes from Sf9 cells expressing AC9, AC6, or β-gal. (B) Dose-response curves of Gαs stimulation of Sf9 membranes expressing AC9 or β-gal in the presence and absence of 100 nM Gβ₁γ₂. Data are shown as means ± S.D. Statistical analyses in (B) were performed with two-way ANOVA followed by a Sidak multiple-comparisons test comparing experimental means of vehicle- and Gαs- or Gβ₁γ₂-stimulated groups at each concentration indicated. n = 3. No statistical difference was found in the presence of Gβγ.

AC9 is not directly regulated by Gαi/o in vitro. (A) Structure-based alignment of amino acids that correspond to the Gαi binding site in AC5. (B) Gαs dose-response curves for AC9 and β-gal Sf9 membranes in the presence and absence of 300 nM Gαi. (C) Gαi dose-response curves for AC9 and β-gal Sf9 membranes in the presence of 300 nM Gαs. (D) E. coli vs. Sf9 purified Gαi isoforms. AC9 Sf9 membranes were stimulated with 300 nM Gαs in the presence or absence of 300 nM Gαi₁ (E. coli or Sf9) or Gαi₃. (E) AC6 membranes with 50 nM Gαs ± 300 nM Gαi proteins served as a positive control. (F) Gαo regulation of AC9 Sf9 membranes stimulated with 300 nM Gαs. (G) AC1 membranes stimulated with 100μM Ca²⁺/300 nM CaM with 1 μM Gαo served as a positive control. Data are shown as means ± S.D. Statistical analyses were performed as follows: (B) and (D), two-way ANOVA followed by a Sidak multiple-comparisons test comparing the vehicle and Gαi/o groups at each concentration indicated; (C), (E), and (F), one-way ANOVA followed by a Tukey multiple-comparisons test comparing experimental means; and (G), t test comparing Ca²⁺/CaM with or without Gαo. For all experiments, n = 3 to 4, performed in duplicate or triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. No statistical difference was found for Gαi/o under any condition with AC9 (B–D and F). ns, not significant.

Neither CaMKII nor PKCβII directly regulates AC9. (A) AC9 membranes were stimulated with 100 nM Gαs in the presence or absence of 200 µM Ca²⁺/1 µM calmodulin (Ca²⁺/CaM), 100 ng CaMKII, or Ca²⁺/CaM plus CaMKII. The inset shows ³²P labeling of MBP by CaMKII, performed in duplicate. As a positive control, AC3 was stimulated with 100 nM Gαs in the presence of 200 µM Ca²⁺/1 µM CaM in the presence or absence of 100 ng CaMKII. (B) AC2, AC9, and β-gal membranes were stimulated with 100 nM Gαs in the presence or absence of 1 µM PMA or PMA plus purified PKCβII. The inset shows ³²P labeling of MBP by PKCβII, performed in duplicate. Data are shown as means ± S.D. Statistical analyses were performed as follows: (A), two-way ANOVA followed by a Sidak multiple-comparisons test; (B), a paired t test comparing the control and the kinase group; and (C), one-way ANOVA followed by a Tukey multiple-comparisons test. For all experiments, n = 3, performed in duplicate. *P < 0.05; **P < 0.01. ns, not significant.

Regulation of endogenous and overexpressed AC9 in COS-7 cells. (A) Representative traces of cells expressing μOR with or without AC6 or AC9 treated with 1 µM isoproterenol (ISO; black square) ± 10 μM DAMGO. Arrows indicate the addition of drugs (ISO/DAMGO). (B) Quantitation of individual cell traces. Data points were averaged (avg) from the 12- to 14-minute timepoints of ISO (1 µM) and ISO plus 10 μM DAMGO. (C) Western blot of COS-7 whole-cell lysates, confirming expression of endogenous or exogenous AC9 (top: rabbit anti-AC9) and AC6 (bottom). (D) Confirmation of endogenous AC9 protein by Western blot of membranes isolated from COS-7 cells, or cells expressing an AC9 siRNA or control siRNA. The Na-K ATPase served as a loading control for COS-7 membranes. Data are shown as means ± S.D. Statistical analyses in (B) were performed with two-way ANOVA followed by a Sidak multiple-comparisons test comparing ISO and ISO plus DAMGO. n = 3–5 with experiments performed in duplicate. **P < 0.01. au, arbitrary unit; KD, knockdown; ns, not significant.

Basal AC activity is dependent on AC9 in COS-7 cells and mouse splenocytes. (A) AC assay of membranes from COS-7 cells stably expressing control or AC9 shRNA/siRNA stimulated with 400 nM Gαs or 50 µM forskolin. (B) Whole-cell basal cAMP accumulation was measured in COS-7 cells expressing control shRNA, AC9 shRNA, or overexpressing (OVE) AC9 using a GloSensor bioluminescent cAMP reporter. Cells were pretreated with 1 mM IBMX for 10 minutes. (C) Quantitative real-time PCR of AC isoforms present in spleen from AC9^−/− mice, normalized to wild-type (WT) expression levels (n = 3). (D) AC assay of WT and AC9 knockout (KO) spleen membranes stimulated with 400 nM Gαs or 50 µM forskolin in the presence or absence of the P-site inhibitor, Ara-A (300 nM). (E and F) Whole-cell cAMP accumulation of mouse splenocytes isolated from WT and AC9KO mice treated with 1 mM IBMX ± 1 μM ISO. Data are shown as means ± S.D. Statistical analysis were performed as follows: (A) and (D), two-way ANOVA followed by a Sidak multiple-comparison test [comparisons within Ara-A treatments in (D) were made separately]; (B), (E), and (F), an unpaired t test of means comparing the indicated groups; and (C), one-way ANOVA followed by a Tukey multiple-comparisons test. For all, experiments were performed in duplicate. n = 3 to 4. *P < 0.05; **P < 0.01; ***P < 0.001. KO, knockout; ns, not significant.