Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates direct sensitization of IP3 receptors by cAMP

Interactions between cyclic adenosine monophosphate (cAMP) and Ca2+ are widespread, and for both intracellular messengers, their spatial organization is important. Parathyroid hormone (PTH) stimulates formation of cAMP and sensitizes inositol 1,4,5-trisphosphate receptors (IP3R) to IP3. We show that PTH communicates with IP3R via “cAMP junctions” that allow local delivery of a supramaximal concentration of cAMP to IP3R, directly increasing their sensitivity to IP3. These junctions are robust binary switches that are digitally recruited by increasing concentrations of PTH. Human embryonic kidney cells express several isoforms of adenylyl cyclase (AC) and IP3R, but IP3R2 and AC6 are specifically associated, and inhibition of AC6 or IP3R2 expression by small interfering RNA selectively attenuates potentiation of Ca2+ signals by PTH. We define two modes of cAMP signaling: binary, where cAMP passes directly from AC6 to IP3R2; and analogue, where local gradients of cAMP concentration regulate cAMP effectors more remote from AC. Binary signaling requires localized delivery of cAMP, whereas analogue signaling is more dependent on localized cAMP degradation.

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Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

10.26434/chemrxiv.9209180 ◽

2019 ◽

Author(s):

Bella Grigorenko ◽

Igor Polyakov ◽

Alexander Nemukhin

Keyword(s):

Adenylyl Cyclase ◽

Bond Cleavage ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Md Simulations ◽

Coordination Shell ◽

Energy Profile ◽

One Step ◽

Type V

We report a mechanism of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) conversion by the mammalian type V adenylyl cyclase revealed in molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations. We characterize a set of computationally derived enzyme-substrate (ES) structures showing an important role of coordination shells of magnesium ions in the solvent accessible active site. Several stable six-fold coordination shells of MgA2+ are observed in MD simulations of ES complexes. In the lowest energy ES conformation, the coordination shell of MgA2+ does not include the Oδ1 atom of the conserved Asp440 residue. Starting from this conformation, a one-step reaction mechanism is characterized which includes proton transfer from the ribose O3'H3' group in ATP to Asp440 via a shuttling water molecule and PA-O3A bond cleavage and O3'-PA bond formation. The energy profile of this route is consistent with the observed reaction kinetics. In a higher energy ES conformation, MgA2+ is bound to the Oδ1(Asp440) atom as suggested in the relevant crystal structure of the protein with a substrate analog. The computed energy profile initiated by this ES is characterized by higher energy expenses to complete the reaction. Consistently with experimental data, we show that the Asp440Ala mutant of the enzyme should exhibit a reduced but retained activity. All considered reaction pathways include proton wires from the O3'H3' group via shuttling water molecules.

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Light-Induced Change of Arginine Conformation Modulates the Rate of Adenosine Triphosphate to Cyclic Adenosine Monophosphate Conversion in the Optogenetic System Containing Photoactivated Adenylyl Cyclase

Journal of Chemical Information and Modeling ◽

10.1021/acs.jcim.0c01308 ◽

2021 ◽

Author(s):

Maria G. Khrenova ◽

Anna M. Kulakova ◽

Alexander V. Nemukhin

Keyword(s):

Adenylyl Cyclase ◽

Adenosine Triphosphate ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Photoactivated Adenylyl Cyclase

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Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis

10.1101/2021.08.03.454881 ◽

2021 ◽

Author(s):

Kaley M. Wilburn ◽

Christine R. Montague ◽

Bo Qin ◽

Ashley K. Woods ◽

Melissa S. Love ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Adenylyl Cyclase ◽

Cholesterol Metabolism ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Camp Signaling ◽

Camp Synthesis ◽

Pharmacokinetic Properties ◽

Bacterial Utilization

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3’, 5’-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required for cholesterol metabolism. Finally, in this work the pharmacokinetic properties of Rv1625c agonists are optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a novel role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

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Targeting cAMP/PKA pathway for glycemic control and type 2 diabetes therapy

Journal of Molecular Endocrinology ◽

10.1530/jme-15-0316 ◽

2016 ◽

Vol 57 (2) ◽

pp. R93-R108 ◽

Cited By ~ 49

Author(s):

Haihua Yang ◽

Linghai Yang

Keyword(s):

Type 2 Diabetes ◽

Glucose Homeostasis ◽

Neural Control ◽

Glycogen Synthesis ◽

Cyclic Adenosine Monophosphate ◽

Glucagon Secretion ◽

Adenosine Monophosphate ◽

Dependent Protein ◽

Pka Pathway

In mammals, cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that is usually elicited by binding of hormones and neurotransmitters to G protein-coupled receptors (GPCRs). cAMP exerts many of its physiological effects by activating cAMP-dependent protein kinase (PKA), which in turn phosphorylates and regulates the functions of downstream protein targets including ion channels, enzymes, and transcription factors. cAMP/PKA signaling pathway regulates glucose homeostasis at multiple levels including insulin and glucagon secretion, glucose uptake, glycogen synthesis and breakdown, gluconeogenesis, and neural control of glucose homeostasis. This review summarizes recent genetic and pharmacological studies concerning the regulation of glucose homeostasis by cAMP/PKA in pancreas, liver, skeletal muscle, adipose tissues, and brain. We also discuss the strategies for targeting cAMP/PKA pathway for research and potential therapeutic treatment of type 2 diabetes mellitus (T2D).

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Syndecan-2 induces filopodia and dendritic spine formation via the neurofibromin–PKA–Ena/VASP pathway

The Journal of Cell Biology ◽

10.1083/jcb.200608121 ◽

2007 ◽

Vol 177 (5) ◽

pp. 829-841 ◽

Cited By ~ 94

Author(s):

Yi-Ling Lin ◽

Ya-Ting Lei ◽

Chen-Jei Hong ◽

Yi-Ping Hsueh

Keyword(s):

Hippocampal Neurons ◽

Actin Polymerization ◽

Kinase Inhibitors ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Human Embryonic Kidney Cells ◽

Spine Formation ◽

Downstream Signaling

Syndecan-2 induced filopodia before spinogenesis; therefore, filopodia formation was used here as a model to study the early downstream signaling of syndecan-2 that leads to spinogenesis. Screening using kinase inhibitors indicated that protein kinase A (PKA) is required for syndecan-2–induced filopodia formation in both human embryonic kidney cells and hippocampal neurons. Because neurofibromin, a syndecan-2–binding partner, activates the cyclic adenosine monophosphate pathway, the role of neurofibromin in syndecan-2–induced filopodia formation was investigated by deletion mutant analysis, RNA interference, and dominant-negative mutant. The results showed that neurofibromin mediates the syndecan-2 signal to PKA. Among actin-associated proteins, Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) were predicted as PKA effectors downstream of syndecan-2, as Ena/VASP, which is activated by PKA, induces actin polymerization. Indeed, when the activities of Ena/VASP were blocked, syndecan-2 no longer induced filopodia formation. Finally, in addition to filopodia formation, neurofibromin and Ena/VASP contributed to spinogenesis. This study reveals a novel signaling pathway in which syndecan-2 activates PKA via neurofibromin and PKA consequently phosphorylates Ena/VASP, promoting filopodia and spine formation.

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Molecular Mechanism of Light-Induced Acceleration of the Adenosine Triphosphate Conversion to the Cyclic Adenosine Monophosphate Catalyzed by the Photoactivated Adenylyl Cyclase bPAC

10.26434/chemrxiv.13139723.v1 ◽

2020 ◽

Author(s):

Alexander Nemukhin ◽

Maria Khrenova ◽

Anna M. Kulakova

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Adenylyl Cyclase ◽

Adenosine Triphosphate ◽

Catalytic Domain ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Bluf Domain ◽

Dynamics Simulations ◽

Photoactivated Adenylyl Cyclase

We report the first computational characterization of an optogenetic system composed of two photosensing BLUF (blue light sensor using flavin adenine dinucleotide) domains and two catalytic adenylyl cyclase (AC) domains. Conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi) catalyzed by ACs coupled with excitation in photosensing domains has emerged in the focus of modern optogenetic applications because of the request in photoregulated enzymes to modulate cellular concentrations of signaling messengers. The photoactivated adenylyl cyclase from the soil bacterium Beggiatoa sp. (bPAC) is an important model showing considerable increase of the ATP to cAMP conversion rate in the catalytic domain after the illumination of the BLUF domain. The 1 μs classical molecular dynamics simulations reveal that the activation of the BLUF domain leading to tautomerization of Gln49 in the chromophore binding pocket results in switching of position of the side chain of Arg278 in the active site of AC. Allosteric signal transmission pathways between Gln49 from BLUF and Arg278 from AC were revealed by the dynamical network analysis. The Gibbs energy profiles of the ATP → cAMP + PPi reaction computed using QM(DFT(ωB97X-D3/6-31G**))/MM(CHARMM) molecular dynamics simulations for both Arg278 conformations in AC clarify the reaction mechanism. In the light-activated system, the corresponding arginine conformation stabilizes the pentacoordinated phosphorus of the α-phosphate group in the transition state, thus lowering the activation energy. Simulations of the bPAC system with the Tyr7Phe replacement in BLUF demonstrate occurrence of both arginine conformations in an equal ratio, explaining the experimentally observed intermediate catalytic activity of the bPAC-Y7F variant as compared with the dark and light states of the wild type bPAC.

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Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events

Thrombosis and Haemostasis ◽

10.1055/s-0039-1688788 ◽

2019 ◽

Vol 119 (07) ◽

pp. 1124-1137 ◽

Cited By ~ 3

Author(s):

Joanne C. Clark ◽

Deirdre M. Kavanagh ◽

Stephanie Watson ◽

Jeremy A. Pike ◽

Robert K. Andrews ◽

...

Keyword(s):

Adenylyl Cyclase ◽

G Protein ◽

Platelet Activation ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Adenosine Diphosphate ◽

Receptor Activation ◽

Inhibitory Effect ◽

Human Platelets ◽

Adenosine Receptor Agonist

Background The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation. Objective This article investigates the effect of A2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets. Methods Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI receptor clustering, cAMP, thromboxane B2 (TxB2) and P-selectin exposure. Results NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA2 mimetic, U46619, but not the combination of adenosine diphosphate and collagen. Conclusion NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA2.

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Bicarbonate, carbon dioxide and pH sensing via mammalian bicarbonate-regulated soluble adenylyl cyclase

Interface Focus ◽

10.1098/rsfs.2020.0034 ◽

2021 ◽

Vol 11 (2) ◽

pp. 20200034

Author(s):

Tom Rossetti ◽

Stephanie Jackvony ◽

Jochen Buck ◽

Lonny R. Levin

Keyword(s):

Carbon Dioxide ◽

Adenylyl Cyclase ◽

Ph Sensor ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Signalling Pathways ◽

Ph Sensing ◽

Carbonic Anhydrases ◽

Soluble Adenylyl Cyclase ◽

Ubiquitous Presence

Soluble adenylyl cyclase (sAC; ADCY10) is a bicarbonate (HCO 3 − )-regulated enzyme responsible for the generation of cyclic adenosine monophosphate (cAMP). sAC is distributed throughout the cell and within organelles and, as such, plays a role in numerous cellular signalling pathways. Carbonic anhydrases (CAs) nearly instantaneously equilibrate HCO 3 − , protons and carbon dioxide (CO 2 ); because of the ubiquitous presence of CAs within cells, HCO 3 − -regulated sAC can respond to changes in any of these factors. Thus, sAC can function as a physiological HCO 3 − /CO 2 /pH sensor. Here, we outline examples where we have shown that sAC responds to changes in HCO 3 − , CO 2 or pH to regulate diverse physiological functions.

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Aquaporin-2 membrane targeting: still a conundrum

AJP Renal Physiology ◽

10.1152/ajprenal.00010.2017 ◽

2017 ◽

Vol 312 (4) ◽

pp. F744-F747 ◽

Cited By ~ 21

Author(s):

Emma T. B. Olesen ◽

Robert A. Fenton

Keyword(s):

Collecting Duct ◽

Water Channel ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Peptide Hormone ◽

Apical Plasma Membrane ◽

Crucial Importance ◽

Aquaporin 2

The targeting of the water channel aquaporin-2 (AQP2) to the apical plasma membrane of kidney collecting duct principal cells is regulated mainly by the antidiuretic peptide hormone arginine vasopressin (AVP). This process is of crucial importance for the maintenance of body water homeostasis. In this brief review we assess the role of cyclic adenosine monophosphate (cAMP) and discuss the emerging concept that type 2 AVP receptor (V2R)-mediated AQP2 trafficking is cAMP-independent.

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