Decades after the discovery of the second messenger cyclic AMP, the regulation of multi-domain adenylyl cyclases remains poorly understood. Mammals express nine membrane-bound isoforms which share an identical domain architecture. These mACs act as downstream effectors of G protein-coupled receptors [GPCRs] and are activated by the release of Gα subunits from heterotrimeric G proteins. Bioinformatic analyses reveal isoform-specific conservation of all mAC domains across more than 500 million years of evolution. The membrane domains function as inhibitory or activating receptors, with unsaturated fatty acids as their ligands. Notable, a similar regulatory mechanism was uncovered in a mycobacterial mAC. This perspective contextualizes previous findings and explores emerging questions regarding signaling via lipid ligands in the cAMP generating system. Due to their low water solubility, fatty acid ligands are likely produced locally at cell-cell contact sites, establishing a semi-stable level of tonic signaling. This suggests the existence of an as-yet unknown link between intercellular lipid signaling and the GPCR-cAMP pathways. Since ligand dissociation appears unlikely, lipid-receptor binding may mark receptors for internalization, recycling, and, possibly, degradation. The potential repertoire of mAC regulation is considered in view of the existence of additional highly conserved domains including those at the N- and C-termini.

The biosynthesis of cyclic 3′,5′-adenosine monophosphate (cAMP) by mammalian membrane-bound adenylyl cyclases (mACs) is predominantly regulated by G-protein-coupled receptors (GPCRs). Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here, we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1–7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities in vitro and in vivo. Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5–mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR–Gsα signaling regulating the biosynthesis of cAMP.

Nine mammalian adenylyl cyclases (AC) are pseudoheterodimers with two hexahelical membrane domains, which are isoform-specifically conserved. Previously we proposed that these membrane domains are orphan receptors (https://doi.org/10.7554/eLife.13098; https://doi.org/10.1016/j.cellsig.2020.109538). Lipids extracted from fetal bovine serum at pH 1 inhibited several mAC activities. Guided by a lipidomic analysis we tested glycerophospholipids as potential ligands. Contrary to expectations we surprisingly discovered that 1-stearoyl-2-docosahexaenoyl-phosphatidic acid (SDPA) potentiated Gsα-activated activity of human AC isoform 3 seven-fold. The specificity of fatty acyl esters at glycerol positions 1 and 2 was rather stringent. 1-Stearoyl-2-docosahexaenoyl-phosphatidylserine and 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine significantly potentiated several Gsα-activated mAC isoforms to different extents. SDPA appears not interact with forskolin activation of AC isoform 3. SDPA enhanced Gsα-activated AC activities in membranes from mouse brain cortex. The action of SDPA was reversible. Unexpectedly, SDPA did not affect cAMP generation in HEK293 cells stimulated by isoproterenol, PGE2 and adenosine, virtually excluding a role as an extracellular ligand and, instead, suggesting an intracellular role. In summary, we discovered a new dimension of intracellular AC regulation by chemically defined glycerophospholipids.

Acidic lipid extracts from mouse liver, kidney, heart, brain, and lung inhibited human pseudoheterodimeric adenylyl cyclases (hACs) expressed in HEK293 cells. Using an acidic lipid extract from bovine lung, a combined MS- and bioassay-guided fractionation identified heme b as inhibitor of membrane-bound ACs. IC50 concentrations were 8-12 μM for the hAC isoforms. Hemopexin and bacterial hemophore attenuated heme b inhibition of hAC5. Structurally related compounds, such as hematin, protoporphyrin IX, and biliverdin, were significantly less effective. Monomeric bacterial class III ACs (mycobacterial ACs Rv1625c; Rv3645; Rv1264; cyanobacterial AC CyaG) were inhibited by heme b with similar efficiency. Surprisingly, structurally related chlorophyll a similarly inhibited hAC5. Heme b inhibited isoproterenol-stimulated cAMP accumulation in HEK293 cells. Using cortical membranes from mouse brain hemin efficiently and reversibly inhibited basal and Gsα-stimulated AC activity. The physiological relevance of heme b inhibition of the cAMP generating system in certain pathologies is discussed.

The nine membrane-delimited eukaryotic adenylyl cyclases are pseudoheterodimers with an identical domain order of seven (nine) distinct subdomains. Bioinformatics show that the protein evolved from a monomeric bacterial progenitor by gene duplication and fusion probably in a primordial eukaryotic cell around 1.5 billion years ago. Over a timespan of about 1 billion years, the first fusion product diverged into nine highly distinct pseudoheterodimeric isoforms. The evolutionary diversification ended approximately 0.5 billion years ago because the present isoforms are found in the living fossil coelacanth, a fish. Except for the two catalytic domains, C1 and C2, the mAC isoforms are fully diverged. Yet, within each isoform a high extent of conservation of respective subdomains is found. This applies to the C- and N-termini, a long linker region between the protein halves (C1b), two short cyclase-transducing-elements (CTE) and notably to the two hexahelical membrane domains TM1 and TM2. Except for the membrane anchor all subdomains were previously implicated in regulatory modalities. The bioinformatic results unequivocally indicate that the membrane anchors must possess an important regulatory function specifically tailored for each mAC isoform.

Mammalian adenylate cyclases (ACs) are pseudoheterodimers with dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. We exchanged both membrane anchors of the AC isoform 2 by the quorum-sensing receptor from Vibrio harveyi, CqsS, which has a ligand, Cholera-Autoinducer-1 (CAI-1). In the chimera, AC activity was stimulated by Gsα, CAI-1 had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation. We report that Gsα stimulation of human AC isoforms 2, 3, 5, and 9 expressed in Sf9 cells is inhibited by serum as is AC activity in membranes isolated from rat brain cortex. AC2 activation by forskolin or forskolin/Gsα was similarly inhibited. Obviously, serum contains as yet unidentified factors affecting AC activity. The data establish a linkage in ACs, in which the membrane anchors, as receptors, transduce extracellular signals to the cytosolic catalytic dimer. A mechanistic three state model of AC regulation is presented compatible with all known regulatory inputs into mammalian ACs. The data allow designating the membrane anchors of mammalian ACs as orphan receptors, and establish a new level of AC regulation.

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane-integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra-histidine signature that is universally present in the membrane anchors of bacterial diheme-B succinate-quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme-B protein and carried a binuclear iron-sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme-B in the membrane and loss of AC activity. Heme-B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q(0) or Q(0)H(2)). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q(0) and O-2, or NADH and Q(0)H(2) respectively. We conclude that CyaC-like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.

Nine pseudoheterodimeric mammalian adenylate cyclases possess two dissimilar hexahelical membrane domains (TM1 and TM2), two dissimilar cyclase-transducing-elements (CTEs) and two complementary catalytic domains forming a catalytic dimer (often termed cyclase-homology-domain, CHD). Canonically, these cyclases are regulated by G-proteins which are released upon ligand activation of G-protein-coupled receptors. So far, a biochemical function of the membrane domains beyond anchoring has not been established. For almost 30 years, work in our laboratory was based on the hypothesis that these voluminous membrane domains possess an additional physiological, possibly regulatory function. Over the years, we have generated numerous artificial fusion proteins between the catalytic domains of various bacterial adenylate cyclases which are active as homodimers and the membrane receptor domains of known bacterial signaling proteins such as chemotaxis receptors and quorum-sensors which have known ligands. Here we summarize the current status of our experimental efforts. Taken together, the data allow the conclusion that the hexahelical mammalian membrane anchors as well as similar membrane anchors from bacterial adenylate cyclase congeners are orphan receptors. A search for as yet unknown ligands of membrane-delimited adenylate cyclases is now warranted.

Nine pseudoheterodimeric mammalian adenylate cyclases possess two dissimilar hexahelical membrane domains (TM1 and TM2), two dissimilar cyclase-transducing-elements (CTEs) and two complementary catalytic domains forming a catalytic dimer (often termed cyclase-homology-domain, CHD). Canonically, these cyclases are regulated by G-proteins which are released upon ligand activation of G-protein-coupled receptors. So far, a biochemical function of the membrane domains beyond anchoring has not been established. For almost 30 years, work in our laboratory was based on the hypothesis that these voluminous membrane domains possess an additional physiological, possibly regulatory function. Over the years, we have generated numerous artificial fusion proteins between the catalytic domains of various bacterial adenylate cyclases which are active as homodimers and the membrane receptor domains of known bacterial signaling proteins such as chemotaxis receptors and quorum-sensors which have known ligands. Here we summarize the current status of our experimental efforts. Taken together, the data allow the conclusion that the hexahelical mammalian membrane anchors as well as similar membrane anchors from bacterial adenylate cyclase congeners are orphan receptors. A search for as yet unknown ligands of membrane-delimited adenylate cyclases is now warranted.

Class III adenylate cyclases (ACs) are widespread signaling proteins, which translate diverse intracellular and extracellular stimuli into a uniform intracellular signal. They are typically composed of an N-terminal array of input domains and transducers, followed C-terminally by a catalytic domain, which, as a dimer, generates the second messenger cAMP. The input domains, which receive stimuli, and the transducers, which propagate the signals, are often found in other signaling proteins. The nature of stimuli and the regulatory mechanisms of ACs have been studied experimentally in only a few cases, and even in these, important questions remain open, such as whether eukaryotic ACs regulated by G protein-coupled receptors can also receive stimuli through their own membrane domains. Here we survey the current knowledge on regulation and intramolecular signal propagation in ACs and draw comparisons to other signaling proteins. We highlight the pivotal role of a recently identified cyclase-specific transducer element located N-terminally of many AC catalytic domains, suggesting an intramolecular signaling capacity.

Class III adenylate cyclases (ACs) are widespread signaling proteins, which translate diverse intracellular and extracellular stimuli into a uniform intracellular signal. They are typically composed of an N-terminal array of input domains and transducers, followed C-terminally by a catalytic domain, which, as a dimer, generates the second messenger cAMP. The input domains, which receive stimuli, and the transducers, which propagate the signals, are often found in other signaling proteins. The nature of stimuli and the regulatory mechanisms of ACs have been studied experimentally in only a few cases, and even in these, important questions remain open, such as whether eukaryotic ACs regulated by G protein-coupled receptors can also receive stimuli through their own membrane domains. Here we survey the current knowledge on regulation and intramolecular signal propagation in ACs and draw comparisons to other signaling proteins. We highlight the pivotal role of a recently identified cyclase-specific transducer element located N-terminally of many AC catalytic domains, suggesting an intramolecular signaling capacity.

Adenylate cyclases (ACs) are signaling proteins that produce the second messenger cAMP. Class III ACs comprise four groups (class IIIa-d) of which class IIIa and IIIb ACs have been identified in bacteria and eukary-otes. Many class IIIa ACs are anchored to membranes via hexahelical domains. In eukaryotic ACs, membrane anchors are well conserved, suggesting that this region possesses important functional characteristics that are as yet unknown. To address this question, we replaced the hexahelical membrane anchor of the mycobacterial AC Rv1625c with the hexahelical quorum-sensing receptor from Legionella, LqsS. Using this chimera, we identified a novel 19-amino-acid cyclase transducer element (CTE) located N-terminally to the catalytic domain that links receptor stimulation to effector activation. Coupling of the receptor to the AC was possible at several positions distal to the membrane exit, resulting in stimulatory or inhibitory responses to the ligand Legionella autoinducer-1. In contrast, on the AC effector side functional coupling was only successful when starting with the CTE. Bioinformatics approaches established that distinct CTEs are widely present in class IIIa and IIIb ACs and in vertebrate guanylate cyclases. The data suggest that membrane-delimited receiver domains transduce regulatory signals to the downstream catalytic domains in an engineered AC model system. This may suggest a previously unknown mechanism for cellular cAMP regulation.

The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins.

Adenylate cyclases convert intra- and extracellular stimuli into a second messenger cAMP signal. Many bacterial and most eukaryotic ACs possess membrane anchors with six transmembrane spans. We replaced the anchor of the AC Rv1625c by the quorum-sensing receptor from Vibrio harveyi which has an identical 6TM design and obtained an active, membrane-anchored AC. We show that a canonical class III AC is ligand-regulated in vitro and in vivo. At 10 µM, the cholera-autoinducer CAI-1 stimulates activity 4.8-fold. A sequence based clustering of membrane domains of class III ACs and quorum-sensing receptors established six groups of potential structural and functional similarities. The data support the notion that 6TM AC membrane domains may operate as receptors which directly regulate AC activity as opposed and in addition to the indirect regulation by GPCRs in eukaryotic congeners. This adds a completely novel dimension of potential AC regulation in bacteria and vertebrates.

Volume 193, no. 5, p. [1086–1089][1], 2011. We were informed by Kevin Young and Gang Li (Microbiology 160: 2079–2088, 2014) that strain H41 is not a tolC mutant as we had described. We have sequenced strain H41 and confirm that it is not a tolC mutant. Rather, the observed phenotype is due to a

Available structures of HAMP domains suggest rotation as one potential mechanism in intraprotein signal transduction. It has been proposed that in poly‐HAMP modules the signal sign is inverted with each additional HAMP. We examined signal transduction through the HAMP tandem domain from the phototaxis transducer of the halophilic archaeon Natronomonas pharaonis in membrane‐bound chimeras consisting of the Escherichia coli chemotaxis receptor for serine, Tsr, as an input and the mycobacterial adenylyl cyclase Rv3645 as an output domain, i.e. the basic chimera was ‘Tsr–NpHAMP tandem–Rv3645 cyclase’. Neither of the NpHAMP units alone nor the NpHAMP tandem transduced a serine signal. After five targeted point mutations in the first α‐helix of NpHAMP1, the non‐functional NpHAMP modules combined into a functional HAMP tandem. 1 mm serine significantly inhibited cyclase activity (−35%; IC50 = 30 μm) in disagreement with the structure‐based predictions. Surprisingly, replacement of NpAS11 in the tandem by the respective AS1 from HAMPTsr resulted in signal inversion, i.e. serine activated cyclase (+129%; EC50 = 10 μm). Examination of 48 mutants of AS11 in the HAMP tandem including two residues of a putative N‐terminal control cable identified five residues in NpAS11 which probably define different ground states of the output domain and thus affect the sign of signal output. The data question the predicted HAMP rotation as the predominant mechanism of intraprotein signal transduction and point to as yet unrecognized conformational motions of HAMP domains in intraprotein signaling.

HAMP domains are small protein modules that predominantly operate as signal transducers in bacterial sensor proteins most of which are membrane delimited. The domain organization of such sensors has the HAMPs localized at the intersection between the membrane-anchored input sensor and the cytosolic output machinery. The data summarized here indicate that HAMP modules use a universal signaling language in balancing the communication between diverse membrane-bound input domains and cytosolic output domains that are completely foreign to each other.