Stimulation of mammalian G-protein-responsive adenylyl cyclases by carbon dioxide

Abstract

Carbon dioxide is fundamental to the physiology of all organisms. There is considerable interest in the precise molecular mechanisms that organisms use to directly sense CO(2). Here we demonstrate that a mammalian recombinant G-protein-activated adenylyl cyclase and the related Rv1625c adenylyl cyclase of Mycobacterium tuberculosis are specifically stimulated by CO(2). Stimulation occurred at physiological concentrations of CO(2) through increased k(cat). CO(2) increased the affinity of enzyme for metal co-factor, but contact with metal was not necessary as CO(2) interacted directly with apoenzyme. CO(2) stimulated the activity of both G-protein-regulated adenylyl cyclases and Rv1625c in vivo. Activation of G-protein regulated adenylyl cyclases by CO(2) gave a corresponding increase in cAMP-response element-binding protein (CREB) phosphorylation. Comparison of the responses of the G-protein regulated adenylyl cyclases and the molecularly, and biochemically distinct mammalian soluble adenylyl cyclase revealed that whereas G-protein-regulated enzymes are responsive to CO(2), the soluble adenylyl cyclase is responsive to both CO(2) and bicarbonate ion. We have, thus, identified a signaling enzyme by which eukaryotes can directly detect and respond to fluctuating CO(2).

FIGURE 1.

Rv1625c is stimulated by CO₂. a, alignment of the catalytic domains of Rv1625c, human AC type 7 C1 domain, rat AC type 2 C2 domain, Slr1991 of Synechocystis, and CyaB1 of Anabaena. Numbers denote the amino acid sequence number. Arrows indicate conserved metal binding aspartate residues. The triangle indicates the substrate binding lysine residue, and the circle is the polymorphic D/T of Class IIIa/b ACs. b, ratio of the specific activities of Rv1625c^204–443 when assayed in the presence of 30 mm total C_i or NaCl at various pH values (1.8 μm protein, 200 μm Mn²⁺-ATP, n = 8). The inset shows the percentage of total C_i made up by CO₂ and over the pH range tested. The figure shows specific activity in the presence of 20 mm NaCl (triangles, right-hand axis) and relative stimulation with Ci (squares, left hand axis). c, cAMP produced by Rv1625c^204–443 under conditions of C_i disequilibrium (36 μm Rv1625c^204–443, 0 °C, 10 s, 20 mm CO₂, 20 mm NaHCO₃, 20 mm NaCl, 100 mm Mes, pH 6.5, 200 μm Mn²⁺-ATP, n = 20; *, p < 0.05). The inset shows a representative control experiment demonstrating that the pH was identical in all assays (circles, NaCl; triangles, NaHCO₃; squares,CO₂; arrow, assay start point). d, Rv1625c^204–443 specific activity (n = 6) was plotted against increasing CO₂. The assay mixture contained 433 nm protein and 200 μm Mn²⁺-ATP, pH 6.5. The total salt concentration was adjusted to 30 mm for all data points.

FIGURE 2.

CO₂ binds Rv1625c in vitro and activates in vivo. a, Rv1625c^204–443-specific activity (n = 6) was plotted against increasing Mn²⁺. The assay mixture contained 1.8 μm protein and 200 μm Mn²⁺-ATP, pH 6.5, and 20 mm NaCl (triangles) or 20 mm NaHCO₃ (7.7 mm CO₂, squares). b, recovered CO₂ from a binding assay in the presence of Rv1625c^204–443, bovine serum albumin (BSA), or buffer alone. c, recovered CO₂ from a binding assay in the presence of Slr1991^120–337 wild type (wt), Slr1991^120–337 D137A D181A (Δmetal), BSA, or buffer alone. d, cAMP-dependent lacZ activity in E. coli under control (vector) conditions or in the presence of Rv1625c^204–443 in samples treated with air or 10% (v/v) CO₂ in air (n = 9; *, p < 0.05). The y axis denotes the concentration of ortho-nitrophenol (ONP) in the lacZ assays performed.

FIGURE 3.

Stimulation of a G-protein regulated AC by CO₂in vitro. a, ratio of the specific activities of 1.1 μm 7C₁ and 5.8 μm 2C₂ when assayed in the presence of 20 mm total C_i or NaCl at various pH values (500 μm Mg²⁺-ATP, 7 μm Gα_s, n = 6). The figure shows specific activity in the presence of 20 mm NaCl (triangles; right-hand axis) and relative stimulation with Ci (squares, left-hand axis). b, cAMP produced by 7C₁·2C₂ under conditions of C_i disequilibrium (20 μm 7C₁, 3.2 μm 2C₂, 0 °C, 10 s, 20 mm CO₂/NaHCO₃/NaCl, 100 mm Mes, pH 6.5, 1 mm Mg²⁺-ATP, 100 μm forskolin, n = 6, *, p < 0.05). Control experiments demonstrated that the pH was identical in all assays. c, 7C₁·2C₂ specific activity (n = 9) was plotted against increasing CO₂ at pH 6.5. The assay mixture contained 1.1 μm 7C₁, 5.8 μm 2C₂, 7 μm Gα_s, and 500 μm Mg²⁺-ATP. The total salt concentration was adjusted to 30 mm for all data points. d, 7C₁·2C₂ specific activity (n = 6) was plotted against increasing Mg²⁺. The assay mixture contained 1.1 μm 7C₁, 5.8 μm 2C₂, 7 μm Gα_s, and 500 μm Mg²⁺-ATP, pH 6.5, and 20 mm NaCl (triangles) or 20 mm NaHCO₃ (7.7 mm CO₂; squares).

FIGURE 4.

sAC is activated by CO₂ and . a, 300 ng of sAC_T was assayed at 30 °C for 30 min with 0.8 mm ATP, 5 mm MgCl₂, and 5 mm CaCl₂ and either 20 mm total Ci or NaCl (n = 4). The figure shows specific activity in the presence of 20 mm NaCl (triangles; right-hand axis) and relative stimulation with Ci (squares; left-hand axis). b, 5 μg of sAC_T was assayed at 0 °C for 10 s at pH 6.4 with 0.8 mm ATP, 5 mm MgCl₂, and 5 mm CaCl₂ with either 20 mm CO₂, NaHCO₃, or NaCl (n = 15; *, p < 0.05).

FIGURE 5.

Stimulation of a G-protein regulated AC by CO₂in vivo. a, monitoring of HEK 293T cell pH_i in response to changing CO₂. b, percentage conversion of ATP into cAMP in HEK 293T cells exposed to varying CO₂ under basal conditions (empty bars) or in the presence of 50 nm isoproterenol (filled bars)(n = 12). c, percentage conversion of ATP into cAMP in HEK 293T cells exposed to air (0.03% CO₂; open bars) or 5% (v/v) CO₂ (filled bars) with 5 μm forskolin and 1 μm KH7 (n = 12; *, p < 0.05). d, lower panel shows immunoblot of HEK 293T cell material after treatment with and without isoproterenol at varying CO₂. The upper panel shows the ratio of phospho-CREB:α-tubulin bands from the quantified bands.