AKAP79 Orchestrates a Cyclic AMP Signalosome Adjacent to Orai1 Ca2+ Channels.

To ensure specificity of response, eukaryotic cells often restrict signalling molecules to sub-cellular regions. The Ca2+ nanodomain is a spatially confined signal that arises near open Ca2+ channels. Ca2+ nanodomains near store-operated Orai1 channels stimulate the protein phosphatase calcineurin, which activates the transcription factor NFAT1, and both enzyme and target are initially attached to the plasma membrane through the scaffolding protein AKAP79. Here, we show that a cAMP signalling nexus also forms adjacent to Orai1. Protein kinase A and phosphodiesterase 4, an enzyme that rapidly breaks down cAMP, both associate with AKAP79 and realign close to Orai1 after stimulation. PCR and mass spectrometry failed to show expression of Ca2+-activated adenylyl cyclase 8 in HEK293 cells, whereas the enzyme was observed in neuronal cell lines. FRET and biochemical measurements of bulk cAMP and protein kinase A activity consistently failed to show an increase in adenylyl cyclase activity following even a large rise in cytosolic Ca2+. Furthermore, expression of AKAP79-CUTie, a cAMP FRET sensor tethered to AKAP79, did not report a rise in cAMP after stimulation, despite AKAP79 association with Orai1. Hence, HEK293 cells do not express functional active Ca2+-activated adenylyl cyclases including adenylyl cyclase 8. Our results show that two ancient second messengers are independently generated in nanodomains close to Orai1 Ca2+ channels.

Structural and Functional Determinants of AC8 Trafficking, Targeting and Responsiveness in Lipid Raft Microdomains.

The fidelity of cAMP in controlling numerous cellular functions rests crucially on the precise organization of cAMP microdomains that are sustained by the scaffolding properties of adenylyl cyclase. Earlier studies suggested that AC8 enriches in lipid rafts where it interacts with cytoskeletal elements. However, these are not stable structures and little is known about the dynamics of AC8 secretion and its interactions. The present study addresses the role of the cytoskeleton in maintaining the AC8 microenvironment, particularly in the context of the trafficking route of AC8 and its interaction with caveolin1. Here, biochemical and live-cell imaging approaches expose a complex, dynamic interaction between AC8 and caveolin1 that affects AC8 processing, targeting and responsiveness in plasma membrane lipid rafts. Site-directed mutagenesis and pharmacological approaches reveal that AC8 is processed with complex N-glycans and associates with lipid rafts en route to the plasma membrane. A dynamic picture emerges of the trafficking and interactions of AC8 while travelling to the plasma membrane, which are key to the organization of the AC8 microdomain.

Adenylyl cyclase signalling complexes - Pharmacological challenges and opportunities.

Signalling pathways involving the vital second messanger, cAMP, impact on most significant physiological processes. Unsurprisingly therefore, the activation and regulation of cAMP signalling is tightly controlled within the cell by processes including phosphorylation, the scaffolding of protein signalling complexes and sub-cellular compartmentalisation. This inherent complexity, along with the highly conserved structure of the catalytic sites among the nine membrane-bound adenylyl cyclases, presents significant challenges for efficient inhibition of cAMP signalling. Here, we will describe the biochemistry and cell biology of the family of membrane-bound adenylyl cyclases, their organisation within the cell, and the nature of the cAMP signals that they produce, as a prelude to considering how cAMP signalling might be perturbed. We describe the limitations associated with direct inhibition of adenylyl cyclase activity, and evaluate alternative strategies for more specific targeting of adenylyl cyclase signalling. The inherent complexity in the activation and organisation of adenylyl cyclase activity may actually provide unique opportunities for selectively targeting discrete adenylyl cyclase functions in disease.

Store-operated Ca²âº-entry and adenylyl cyclase.

One of the longest-standing effects of SOCE is in its selective regulation of Ca(2+)-sensitive adenylyl cyclase (AC) activity in non-excitable cells. Remarkably it was this source of Ca(2+) (SOCE) rather than the apparent magnitude of the Ca(2+)-rise that conferred AC responsiveness. The molecular basis for this dependence is now resolved in the case of adenylyl cyclase 8 (AC8). Sensors for Ca(2+) and cAMP targeted to ACs have been particularly useful in dissecting the influences upon and composition of what turn out to be signalling microdomains centred on ACs. A number of physiological processes depend on the regulation by SOCE of ACs, but the issue is under-studied. Here I will expand on these topics and point to some immediate unresolved questions.

Adenylate cyclase-centred microdomains.

Recent advances in the AC (adenylate cyclase)/cAMP field reveal overarching roles for the ACs. Whereas few processes are unaffected by cAMP in eukaryotes, ranging from the rapid modulation of ion channel kinetics to the slowest developmental effects, the large number of cellular processes modulated by only three intermediaries, i.e. PKA (protein kinase A), Epacs (exchange proteins directly activated by cAMP) and CNG (cyclic nucleotide-gated) channels, poses the question of how selectivity and fine control is achieved by cAMP. One answer rests on the number of differently regulated and distinctly expressed AC species. Specific ACs are implicated in processes such as insulin secretion, immunological responses, sino-atrial node pulsatility and memory formation, and specific ACs are linked with particular diseased conditions or predispositions, such as cystic fibrosis, Type 2 diabetes and dysrhythmias. However, much of the selectivity and control exerted by cAMP lies in the sophisticated properties of individual ACs, in terms of their coincident responsiveness, dynamic protein scaffolding and organization of cellular microassemblies. The ACs appear to be the centre of highly organized microdomains, where both cAMP and Ca2+, the other major influence on ACs, change in patterns quite discrete from the broad cellular milieu. How these microdomains are organized is beginning to become clear, so that ACs may now be viewed as fundamental signalling centres, whose properties exceed their production of cAMP. In the present review, we summarize how ACs are multiply regulated and the steps that are put in place to ensure discrimination in their signalling. This includes scaffolding of targets and modulators by the ACs and assembling of signalling nexuses in discrete cellular domains. We also stress how these assemblies are cell-specific, context-specific and dynamic, and may be best addressed by targeted biosensors. These perspectives on the organization of ACs uncover new strategies for intervention in systems mediated by cAMP, which promise far more informed specificity than traditional approaches.

TRPC1 contributes to the Ca2+-dependent regulation of adenylate cyclases.

SOCE (store-operated Ca2+ entry) is mediated via specific plasma membrane channels in response to ER (endoplasmic reticulum) Ca2+ store depletion. This route of Ca2+ entry is central to the dynamic interplay between Ca2+ and cAMP signalling in regulating the activity of Ca2+-sensitive adenylate cyclase isoforms (AC1, AC5, AC6 and AC8). Two proteins have been identified as key components of SOCE: STIM1 (stromal interaction molecule 1), which senses ER Ca2+ store content and translocates to the plasma membrane upon store depletion, where it then activates Orai1, the pore-forming component of the CRAC (Ca2+ release-activated Ca2+) channel. Previous studies reported that co-expression of STIM1 and Orai1 in HEK-293 (human embryonic kidney 293) cells enhances Ca2+-stimulated AC8 activity and that AC8 and Orai1 directly interact to enhance this regulation. Nonetheless, the additional involvement of TRPC (transient receptor potential canonical) channels in SOCE has also been proposed. In the present study, we evaluate the contribution of TRPC1 to SOCE-mediated regulation of Ca2+-sensitive ACs in HEK-293 cells stably expressing AC8 (HEK-AC8) and HSG (human submandibular gland) cells expressing an endogenous Ca2+-inhibited AC6. We demonstrate a role for TRPC1 as an integral component of SOCE, alongside STIM1 and Orai1, in regulating Ca2+ fluxes within AC microdomains and influencing cAMP production.

High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application.

ADCY5 couples glucose to insulin secretion in human islets.

Single nucleotide polymorphisms (SNPs) within the ADCY5 gene, encoding adenylate cyclase 5, are associated with elevated fasting glucose and increased type 2 diabetes (T2D) risk. Despite this, the mechanisms underlying the effects of these polymorphic variants at the level of pancreatic ß-cells remain unclear. Here, we show firstly that ADCY5 mRNA expression in islets is lowered by the possession of risk alleles at rs11708067. Next, we demonstrate that ADCY5 is indispensable for coupling glucose, but not GLP-1, to insulin secretion in human islets. Assessed by in situ imaging of recombinant probes, ADCY5 silencing impaired glucose-induced cAMP increases and blocked glucose metabolism toward ATP at concentrations of the sugar >8 mmol/L. However, calcium transient generation and functional connectivity between individual human ß-cells were sharply inhibited at all glucose concentrations tested, implying additional, metabolism-independent roles for ADCY5. In contrast, calcium rises were unaffected in ADCY5-depleted islets exposed to GLP-1. Alterations in ß-cell ADCY5 expression and impaired glucose signaling thus provide a likely route through which ADCY5 gene polymorphisms influence fasting glucose levels and T2D risk, while exerting more minor effects on incretin action.

An improved targeted cAMP sensor to study the regulation of adenylyl cyclase 8 by Ca2+ entry through voltage-gated channels.

Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic ß-cell line. In these cells, AC8 was activated by Ca(2+) entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca(2+) channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes.

Structural insights into calmodulin/adenylyl cyclase 8 interaction.

Calmodulin (CaM) is a highly conserved intracellular Ca(2+)-binding protein that exerts important functions in many cellular processes. Prominent examples of CaM-regulated proteins are adenylyl cyclases (ACs), which synthesize cAMP as a central second messenger. The interaction of ACs with CaM represents the link between Ca(2+)-signaling and cAMP-signaling pathways. Thereby, different AC isoforms stimulated by CaM, comprise diverse mechanisms of regulation by the Ca(2+) sensor. To extend the structural information about the detailed mechanisms underlying the regulation of AC8 by CaM, we employed an integrated approach combining chemical cross-linking and mass spectrometry with two peptides representing the CaM-binding regions of AC8. These experiments reveal that the structures of CaM/AC8 peptide complexes are similar to that of the CaM/skeletal muscle myosin light chain kinase peptide complex where CaM is collapsed around the target peptide that binds to CaM in an antiparallel orientation. Cross-linking experiments were complemented by investigating the binding of AC8 peptides to CaM thermodynamically with isothermal titration calorimetry. There were no hints on a complex, in which both AC8 peptides bind simultaneously to CaM, refining our current understanding of the interaction between CaM and AC8.

AKAP79, PKC, PKA and PDE4 participate in a Gq-linked muscarinic receptor and adenylate cyclase 2 cAMP signalling complex.

AC2 (adenylate cyclase 2) is stimulated by activation of Gq-coupled muscarinic receptors through PKC (protein kinase C) to generate localized cAMP in HEK (human embryonic kidney)-293 cells. In the present study, we utilized a sensitive live-cell imaging technique to unravel the proteins that play essential roles in a Gq-coupled muscarinic receptor-mediated cAMP signalling complex. We reveal that, upon agonist binding to the Gq-coupled muscarinic receptor, AKAP79 (A-kinase-anchoring protein 79) recruits PKC to activate AC2 to produce cAMP. The cAMP formed is degraded by PDE4 (phosphodiesterase 4) activated by an AKAP-anchored PKA (protein kinase A). Calcineurin, a phosphatase bound to AKAP79, is not involved in this regulation. Overall, a transient cAMP increase is generated from AC2 by Gq-coupled muscarinic receptor activation, subject to sophisticated regulation through AKAP79, PKC, PDE4 and PKA, which significantly enhances acetylcholine-mediated signalling.

Distinct mechanisms of calmodulin binding and regulation of adenylyl cyclases 1 and 8.

Calmodulin (CaM), by mediating the stimulation of the activity of two adenylyl cyclases (ACs), plays a key role in integrating the cAMP and Ca(2+) signaling systems. These ACs, AC1 and AC8, by decoding discrete Ca(2+) signals can contribute to fine-tuning intracellular cAMP dynamics, particularly in neurons where they predominate. CaM comprises an α-helical linker separating two globular regions at the N-terminus and the C-terminus that each bind two Ca(2+) ions. These two lobes have differing affinities for Ca(2+), and they can interact with target proteins independently. This study explores previous indications that the two lobes of CaM can regulate AC1 and AC8 differently and thereby yield different responses to cellular transitions in [Ca(2+)](i). We first compared by glutathione S-transferase pull-down assays and offline nanoelectrospray ionization mass spectrometry the interaction of CaM and Ca(2+)-binding deficient mutants of CaM with the internal CaM binding domain (CaMBD) of AC1 and the two terminal CaMBDs of AC8. We then examined the influence of these three CaMBDs on Ca(2+) binding by native and mutated CaM in stopped-flow experiments to quantify their interactions. The three CaMBDs show quite distinct interactions with the two lobes of CaM. These findings establish the critical kinetic differences between the mechanisms of Ca(2+)-CaM activation of AC1 and AC8, which may underpin their different physiological roles.

A key phosphorylation site in AC8 mediates regulation of Ca(2+)-dependent cAMP dynamics by an AC8-AKAP79-PKA signalling complex.

Adenylyl cyclase (AC) isoforms can participate in multimolecular signalling complexes incorporating A-kinase anchoring proteins (AKAPs). We recently identified a direct interaction between Ca(2+)-sensitive AC8 and plasma membrane-targeted AKAP79/150 (in cultured pancreatic insulin-secreting cells and hippocampal neurons), which attenuated the stimulation of AC8 by Ca(2+) entry (Willoughby et al., 2010). Here, we reveal that AKAP79 recruits cAMP-dependent protein kinase (PKA) to mediate the regulatory effects of AKAP79 on AC8 activity. Modulation by PKA is a novel means of AC8 regulation, which may modulate or apply negative feedback to the stimulation of AC8 by Ca(2+) entry. We show that the actions of PKA are not mediated indirectly via PKA-dependent activation of protein phosphatase 2A (PP2A) B56δ subunits that associate with the N-terminus of AC8. By site-directed mutagenesis we identify Ser-112 as an essential residue for direct PKA phosphorylation of AC8 (Ser-112 lies within the N-terminus of AC8, close to the site of AKAP79 association). During a series of experimentally imposed Ca(2+) oscillations, AKAP79-targeted PKA reduced the on-rate of cAMP production in wild-type but not non-phosphorylatable mutants of AC8, which suggests that the protein-protein interaction may provide a feedback mechanism to dampen the downstream consequences of AC8 activation evoked by bursts of Ca(2+) activity. This fine-tuning of Ca(2+)-dependent cAMP dynamics by targeted PKA could be highly significant for cellular events that depend on the interplay of Ca(2+) and cAMP, such as pulsatile hormone secretion and memory formation.

Muscarinic receptors stimulate AC2 by novel phosphorylation sites, whereas Gßγ subunits exert opposing effects depending on the G-protein source.

Direct phosphorylation of AC2 (adenylyl cyclase 2) by PKC (protein kinase C) affords an opportunity for AC2 to integrate signals from non-canonical pathways to produce the second messenger, cyclic AMP. The present study shows that stimulation of AC2 by pharmacological activation of PKC or muscarinic receptor activation is primarily the result of phosphorylation of Ser490 and Ser543, as opposed to the previously proposed Thr1057. A double phosphorylation-deficient mutant (S490/543A) of AC2 was insensitive to PMA (phorbol myristic acid) and CCh (carbachol) stimulation, whereas a double phosphomimetic mutant (S490/543D) mimicked the activity of PKC-activated AC2. Putative Gßγ-interacting sites are in the immediate environment of these PKC phosphorylation sites (Ser490 and Ser543) that are located within the C1b domain of AC2, suggesting a significant regulatory importance of this domain. Consequently, we examined the effect of both Gq-coupled muscarinic and Gi-coupled somatostatin receptors. Employing pharmacological and FRET (fluorescence resonance energy transfer)-based real-time single cell imaging approaches, we found that Gßγ released from the Gq-coupled muscarinic receptor or Gi-coupled somatostatin receptors exert inhibitory or stimulatory effects respectively. These results underline the sophisticated regulatory capacities of AC2, in not only being subject to regulation by PKC, but also and in an opposite manner to Gßγ subunits, depending on their source.

Direct binding between Orai1 and AC8 mediates dynamic interplay between Ca2+ and cAMP signaling.

The interplay between calcium ion (Ca(2+)) and cyclic adenosine monophosphate (cAMP) signaling underlies crucial aspects of cell homeostasis. The membrane-bound Ca(2+)-regulated adenylyl cyclases (ACs) are pivotal points of this integration. These enzymes display high selectivity for Ca(2+) entry arising from the activation of store-operated Ca(2+) (SOC) channels, and they have been proposed to functionally colocalize with SOC channels to reinforce crosstalk between the two signaling pathways. Using a multidisciplinary approach, we have identified a direct interaction between the amino termini of Ca(2+)-stimulated AC8 and Orai1, the pore component of SOC channels. High-resolution biosensors targeted to the AC8 and Orai1 microdomains revealed that this protein-protein interaction is responsible for coordinating subcellular changes in both Ca(2+) and cAMP. The demonstration that Orai1 functions as an integral component of a highly organized signaling complex to coordinate Ca(2+) and cAMP signals underscores how SOC channels can be recruited to maximize the efficiency of the interplay between these two ubiquitous signaling pathways.

Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu.

The central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane; an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts; the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub.

cAMP measurements with FRET-based sensors in excitable cells.

The development of FRET (fluorescence resonance energy transfer)-based sensors for measuring cAMP has opened the door to sophisticated insights into single-cell cAMP dynamics. cAMP can be measured in distinct cell populations and even in distinct microdomains within cells. However, there is still only limited information on cAMP dynamics in excitable cells, particularly as a function of the activity of voltage-gated Ca2+ channels. A major reason for this is the pH shifts that can occur in excitable cells and their effects on fluorescent proteins.

Palmitoylation targets AKAP79 protein to lipid rafts and promotes its regulation of calcium-sensitive adenylyl cyclase type 8.

PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca(2+) entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.