Molecular details of cAMP generation in mammalian cells: a tale of two systems

Abstract

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.

Figure 1

Revised model for cAMP signaling. The second messenger is formed, acts, and is degraded close to its target, leading to the formation of cAMP micro-domains within the cell. H denotes a receptor-activating hormone and broken lines indicate the limited diffusion sphere of cAMP formed by the respective adenylyl cyclase.

Figure 2

Scheme of domain arrangements in various class III cyclases. The conserved, dimeric catalytic core is formed by association of two domains, which belong either to a single polypeptide or to two protein chains. Intermolecular dimers can be homodimers or heterodimers. The dimers contain either two active sites (homodimers; indicated by circles) or one active site and a degenerate, inactive site (broken circle in heterodimers and pseudoheterodimers). C, catalytic domain; TM, transmembrane domain.

Figure 3

Phylogenetic relationship between various class III ACs and GCs. Amino acid sequences of the catalytic regions were aligned using ClustalW and represented as an unrooted tree using Fitch (Phylip 3.5; Felsenstein, J., Department of Genetics, University of Washington, Seattle) using E. coli CyaA as an outgroup. Numbers represent bootstrap confidence values of 1000 replicates. The individual catalytic domains from cyclases with two catalytic domains, i.e. C₁ and C₂, on a single polypeptide chain were analyzed separately. Accession numbers for the aligned amino acid sequences are as follows: human ANP-A (hGCA), P16066; human ANP-B (hGCB), P26594; human sGCα2 (hGCa2), P33402; human sGCα3 (hGCa3), Q02108; human sGCβ1 (hGCb1), Q02153; human sGCβ2 (hGCb2), O75343; human sGCβ3 (hGCb3), NP_000848; human GC-2C (hGCCC), P25092; human GC-2D (hGCD), Q02846; human GC-2F (hGCF), P51841; Plasmodium falciparum GCα (PfGCaC1/C2), NP_705488; P. falciparum GCβ (PfGCbC1/C2), AAN35978; Dictyostelium discoideum gca (DdGcaC1/C2), XP_643824; D. discoideum SgcA (DdSgcAC1/C2), XP_643219; Drosophila melanogaster Gyc32E (DmGC32E), Q07553; D. melanogaster Gyc88E (DmGC88E), NP_731974; D. melanogaster CG31183 (CG31183), NP_650505; D. melanogaster CG10738 (CG10738), NP_729905; D. melanogaster CG9783 (CG9783), NP_649477; D. melanogaster CG3216 (CG3216), NP_611532; D. melanogaster 99B (DmGC99B), Q07093; human tmAC-1 (htmAC1C1/ C2), Q08828; human tmAC-2 (htmAC2C1/C2), Q08462; human tmAC-3 (htmAC3C1/C2), O60266; human tmAC-4 (htmAC4C1/C2), Q8NFM4; human tmAC-5 (htmAC5C1/C2), O95622; human tmAC-6 (htmAC6C1/C2), O43306; human tmAC-7 (htmAC7C1/C2), P51828; human tmAC-8 (htmAC8C1/C2), P40145; human tmAC-9 (htmAC9C1/C2), O60503; D. melanogaster AC-A (DmACAC1/C2), NP_620475; D. melanogaster AC-B (DmACBC1/C2), NP_620474; D. melanogaster AC-C (DmACCC1/C2), NP_609593; D. melanogaster AC-D (DmACDC1/C2), NP_620478; D. melanogaster AC-E (DmACEC1/C2), NP_620479; D. melanogaster AC-1 (DmAC1C1/C2); Anopheles gambiae AgaC (AgACC1/ C2), EAA10271; human sAC (hsACC1/C2), NP_060887; P. falciparum ACα (PfAca), NP_701931; P. falciparum ACβ (PfACbC1/C2), NP_704518; D. discoideum Aca (DdAcaC1/C2), AAA33163; D. discoideum AcaA (DdAcaAC1/C2), XP_640636; D. discoideum AcrA (DdAcrA), AAD50121; D. discoideum AcgA (DdAcg), Q03101; Trypanosoma brucei AC-1 (TbbAC1), Q99279; T. brucei AC-2 (TbbAC2), Q99396; T. brucei AC-3 (TbbAC3), Q99280; T. brucei AC-4 (TbbAC4), Q26721; T. congolense AC (TcongAC), Q26896; T. equiperdum AC (TequiAC), P26338; Mycobacterium tuberculosis Rv1264 (Rv1624), CAB00890; M. tuberculosis Rv1319c (Rv1319c), Q10632; M. tuberculosis Rv1625c (Rv1625c), P0A4Y0; Candida albicans Cyr1 (CalbAC), AAG18428; Cryptococcus neoformans AC (CneoACC1/C2), AAG60619; Schizosaccharomyces pombe AC (SpomACC1/C2), P14605; Saccharomyces cerevisiae AC (ScerACC1/C2), P08678; Chloroflexus aurantiacus Chlo1066 (Ch1066C1/C2), ZP_00018085; C. aurantiacus Chlo1187 (Ch1187C1/C2), ZP_00018205; C. aurantiacus Chlo1431 (Ch1431C1/C2), ZP_00018442; Spirulina platensis CyaA (SplatACA), BAA22996; S. platensis CyaC (SplatACC), T17197; Stigmatella aurantiaca CyaA (SaurACA), CAA11549; S. aurantiaca CyaB1 (SaurACB1), T10905; Synecocystis sp. CyaA2 (PCC6803), BAA16969; Anabaena pirulensis CyaA (ApirACA), P43524; A. pirulensis CyaB1 (ApirACB1), NP_486306; A. pirulensis CyaB2 (ApirACB2), BAA13999; A. pirulensis CyaC (ApirACC), BAA14000; Mycobacterium leprae AC (MlepAC), CAA19149; Sinorhizobium melioti AC-1 (SmeliAC1), P19485; S. melioti AC-2 (SmeliAC2), Q52915; S. melioti AC-3 (SmeliAC3), Q9Z3Q0; Mesorhizobium loti AC-3 (MlotiAC3), BAB50205; Myxococcus xanthus CyaA (MxanCyaA), BAC00918; M. xanthus CyaB (MxanCyaB), BAD98264; Rhizobium etli CyaA (RetlCyaA), AAM66143; R. etli CyaB (RetlCyaB), AAM66145; Anenome cylindrica A (AcylACA), P43524; E. coli ACA (EcoliACA), CAA47280.

Figure 4

Structure of class III catalytic units. (a) Overall structures of the catalytic domains of a mammalian tmAC (left) and the cyanobacterial sAC-like enzyme CyaC (right), both in complex with ATP analogs. The two subunits of the catalytic cores are colored red and blue, respectively, and secondary structure elements are labeled according to Steegborn et al., and Tesmer et al. (b) Active site of the sAC-like cyclase CyaC in complex with the ATP analog Rp-ATPαS and two magnesium ions A and B (yellow spheres). The ion coordinating Asp residues 1017 and 1061, the residues recognizing the adenine ring (1139* and 1057*), the phosphate binding Arg (1150*), and the Ala conserved in sACs and replaced by Ser in tmACs (1149*) are labeled. This Figure was generated with PyMol.

Figure 5

Structure-based sequence alignment of representative members of nucleotidyl cyclase class III. Included are bicarbonate responsive cyclases (top 9 sequences) from bacteria, fungi, and human; the human G protein-regulated tmACs II and V and the structurally characterized tmACs from rat and dog; and two guanylyl cyclases, the homodimeric transmembrane receptor GC-A and the heterodimeric soluble guanylyl cyclases sGC1 (bottom three sequences). Secondary structure elements of CyaC are shown on top and those of tmAC II C₂ at the bottom. Ion binding residues (▽) and residues binding the substrate (○) or the transition state (◁) are labeled with filled and empty symbols indicating C₁ and C₂ residues, respectively. CyaC Thr1139*, which is characteristic for sAC enzymes and replaced by Asp in tmACs and Cys in GCs, is indicated (△). Homologous regions are shaded red, and highly conserved positions are shown in blue. Residue numbering on the top refers to CyaC and at the bottom to human tmAC VC₁ and IIC₂, respectively. The Figure was generated with Alscript.

Figure 6

Catalytic mechanism of class III AC enzymes. Schematic view of the two-ion catalyzed reaction, which starts with the substrate ATP and yields the products cAMP and pyrophosphate. The reaction proceeds through a magnesium-stabilized penta-covalent transition state with an in-line arrangement of the incoming and the leaving group.

Figure 7

Modulation of class III AC catalytic activity. (a) Complex of the tmAC catalytic core with G_sα. Displayed is an overlay of the tmAC/G_sα complex in the absence (grey) and in the presence (red) of the ATP analog Rp–ATPαS, showing the large movement of α1 upon ligand binding, accompanied by a smaller shift of the β7/β8 loop. (b) Active site of a tmAC occupied by two Mg²⁺ (yellow spheres) and the inhibitor MANT–GTP. The MANT fluorophor extends from the active site into the dimer interface patch formed by α1 and the β7/β8 loop, the structural elements, which are responsible for the open–closed transition of the enzyme. (c) Bicarbonate inducible closure of the sAC active site. Overlay of the sAC–α,β-Me-ATP structure (open state; darkest gray, α1 and β7–β8 in blue), the sAC–Rp-ATPαS complex (partially closed; middle gray and red), and the bicarbonate-soaked Rp-ATPαS structure (closed; lightest gray and yellow). This Figure was reproduced from Steegborn et al. (d) Active site region of CyaC in complex with the substrate analog α,β-Me-ATP and the non-competitive inhibitor catechol estrogen (CE). The inhibitor is bound next to the active site and acts as chelator on the catalytic magnesium ion (yellow sphere), thereby distorting the active site and the substrate analog (green sphere: calcium). Two inhibitor molecules are bound due to the symmetry of the homodimeric sAC homolog CyaC used in this study. This Figure was generated with PyMol and Setor (c).