AC types 1, 2, 3, and 9 associate with Yotiao. (A) Immunoprecipitations of V5-tagged Yotiao from transfected HEK293 cells were assayed for AC activity. Samples transfected with Yotiao plus AC 1 (P < 0.05), 2 (P < 0.01), or 3 (P < 0.05) had significant AC activity compared with the AC isoform alone. Yotiao expression was confirmed by Western blot analysis. (B) Cell lysates (5% of starting material used in A) were assayed for AC activity to confirm expression of each AC isoform over vector alone. (C) Cells transfected with AC 9 and Yotiao were immunoprecipitated with α-Yotiao serum and then stimulated for associated AC activity with 400 nM Gαs (P < 0.05). (D) AC activity in transfected cell lysates used in C. (E) Membranes from HEK293 cells transfected with vector, or AC2 +/− Yotiao, were stimulated with either 50 nM Gαs (330 pmol/min/mg; P < 0.001), 50 nM Gαs and 30 nM Gβγ (1,606 pmol/min/mg; P < 0.001), or 100 μM forskolin (131 pmol/min/mg; P < 0.001). Western blot analysis of AC2 expression levels is shown below. (F) Membranes expressing AC3 were inhibited by Yotiao when stimulated with 50 nM Gαs and 100 μM forskolin (P < 0.01) but not Gαs alone. Western blot analysis confirmed that AC3 expression was not altered by Yotiao.

Amino acids 808–957 of Yotiao are required for AC2 binding and inhibition, and compete for AC2–Yotiao interactions. (A) Diagram of Yotiao fragments, each containing cMYC tags. (B and C) Yotiao fragments were expressed in HEK293 cells and subjected to GST pull-down assay. Deletion of 808–966 of Yotiao (ΔFL) results in loss of Yotiao binding to GST–AC2-NT. (D) GST–AC2-NT binds specifically and directly to E. coli purified 808–957 (C*) but not to 953–1171 (D*). (E) IP-AC assay of AC2 association with Yotiao and fragments of Yotiao. Yotiao and corresponding fragments, shown in A, were expressed in HEK293 cells with AC2 and immunoprecipitated with mouse α-cMYC before stimulation with 50 nM Gαs and 30 nM Gβγ (P < 0.001, for Yotiao A, B, and C fragments). (F) Membranes from transfected HEK293 cells were stimulated with Gαs and Gβγ. YotiaoΔ808–966 (ΔFL) had no effect on AC2 activity, and full length Yotiao inhibited AC2 by ∼50% (P < 0.001). (G) Membranes from HEK293 cells transfected with AC2 +/− Yotiao were incubated with 5 μM purified Yotiao 808–957 or 953–1171 before stimulation with Gαs and Gβγ. Yotiao 808–957 reversed the inhibition of AC2 by Yotiao, whereas 953–1171 had no effect (P < 0.001). (H) Rat brain membranes were incubated with buffer and purified Yotiao 808–957 or 953–1171 (5 μM) before stimulation with 50 nM Gαs and 30 nM Gβγ. Membranes incubated with purified Yotiao 808–957 had 38 ± 2.9% (P < 0.001) more AC activity than membranes incubated with buffer or 953–1171.

AC associates with Yotiao in brain and heart. (A) Western blot of HEK293 cell extracts expressing +/− Yotiao using Yotiao anti-serum. (B and C) Yotiao was immunoprecipitated from (B) rat brain or (C) rat heart extracts by using Yotiao anti-serum or preimmune serum. Immunoprecipitants were stimulated with 50 nM Gαs-GTPγS (Gαs) and 100 μM forskolin to measure associated AC activity. Significantly more AC activity associates with α-Yotiao in brain (P < 0.001) and heart (P < 0.01) compared with preimmune serum.

AC2 N terminus (AC2-NT) binds Yotiao and reverses Yotiao inhibition of AC2. (A) Extracts from cells expressing vector or Yotiao were incubated with GST or GST–AC2-NT in a GST pull-down assay. Western blot analysis indicates an interaction of Yotiao with GST–AC2-NT (Upper). GST protein input is shown (Lower). (B and C) Membranes from transfected HEK293 cells were incubated with either GST or GST–AC2-NT (5 μM) for 15 min on ice before stimulation with 50 nM Gαs and 30 nM Gβγ for AC2 (B) or 50 nM Gαs and 100 μM forskolin for AC3 (C). Addition of AC2-NT reverses inhibition of AC2 activity by Yotiao (P < 0.001), but not that of AC3 (P < 0.001). (D) Transfected HEK293 cell lysates were incubated with GST or GST–AC2-NT (5 μM) before immunoprecipitation with α-Yotiao serum. Immunoprecipitants were assayed for associated AC activity with Gαs and Gβγ (P < 0.01). (E) Rat brain extracts were incubated with GST or GST–AC2-NT before immunoprecipitation and assay of AC activity with Gαs and forskolin (*, GST, P < 0.05; **, AC2-NT, P < 0.01). GST–AC2-NT blocked AC association with Yotiao.