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.

Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells.

Microdomains have been proposed to explain specificity in the myriad of possible cellular targets of cAMP. Local differences in cAMP levels can be generated by phosphodiesterases, which control the diffusion of cAMP. Here, we address the possibility that adenylyl cyclases, the source of cAMP, can be primary architects of such microdomains. Distinctly regulated adenylyl cyclases often contribute to total cAMP levels in endogenous cellular settings, making it virtually impossible to determine the contribution of a specific isoform. To investigate cAMP dynamics with high precision at the single-isoform level, we developed a targeted version of Epac2-camps, a cAMP sensor, in which the sensor was tagged to a catalytically inactive version of the Ca(2+)-stimulable adenylyl cyclase 8 (AC8). This sensor, and less stringently targeted versions of Epac2-camps, revealed opposite regulation of cAMP synthesis in response to Ca(2+) in GH(3)B(6) pituitary cells. Ca(2+) release triggered by thyrotropin-releasing hormone stimulated the minor endogenous AC8 species. cAMP levels were decreased by inhibition of AC5 and AC6, and simultaneous activation of phosphodiesterases, in different compartments of the same cell. These findings demonstrate the existence of distinct adenylyl-cyclase-centered cAMP microdomains in live cells and open the door to their molecular micro-dissection.

AKAP79/150 interacts with AC8 and regulates Ca2+-dependent cAMP synthesis in pancreatic and neuronal systems.

Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca(2+). However, whether AKAPs play a role in the control of AC activity by Ca(2+) is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca(2+)-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca(2+) events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca(2+)-stimulated cAMP production.

Capacitative Ca2+ entry via Orai1 and stromal interacting molecule 1 (STIM1) regulates adenylyl cyclase type 8.

Capacitative Ca(2+) entry (CCE), which occurs through the plasma membrane as a result of Ca(2+) store depletion, is mediated by stromal interacting molecule 1 (STIM1), a sensor of intracellular Ca(2+) store content, and the pore-forming component Orai1. However, additional factors, such as C-type transient receptor potential (TRPC) channels, may also participate in the CCE apparatus. To explore whether the store-dependent Ca(2+) entry reconstituted by coexpression of Orai1 and STIM1 has the functional properties of CCE, we used the Ca(2+)-calmodulin stimulated adenylyl cyclase type 8 (AC8), which responds selectively to CCE, whereas other modes of Ca(2+) entry, including those activated by arachidonate and the ionophore ionomycin, are ineffective. In addition, the Ca(2+) entry mediated by previous CCE candidates, diacylglycerol-activated TRPC channels, does not activate AC8. Here, we expressed Orai1 and STIM1 in HEK293 cells and saw a robust increment in CCE, and a proportional increase in CCE-stimulated AC8 activity. Inhibitors of the CCE assembly process ablated the effects on cyclase activity in both AC8-overexpressing HEK293 cells and insulin-secreting MIN6 cells endogenously expressing Ca(2+)-sensitive AC isoforms. AC8 is believed to be closely associated with the source of CCE; indeed, not only were AC8, Orai1, and STIM1 colocalized at the plasma membrane but also all three proteins occurred in lipid rafts. Together, our data indicate that Orai1 and STIM1 can be integral components of the cAMP and CCE microdomain associated with adenylyl cyclase type 8.

Kinetic properties of Ca2+/calmodulin-dependent phosphodiesterase isoforms dictate intracellular cAMP dynamics in response to elevation of cytosolic Ca2+.

Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca2+-sensitive ACs have received far greater attention than the Ca2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca2+ concentration ([Ca2+]i). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater Km for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca2+]i and thereby provide a rationale for the utility of diverse PDE1 species.

Distinct mechanisms of regulation by Ca2+/calmodulin of type 1 and 8 adenylyl cyclases support their different physiological roles.

Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca(2+)/calmodulin (CaM), have discrete neuronal functions. Although the Ca(2+) sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca(2+)/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca(2+)/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca(2+)/CaM both in vivo and in vitro and explore the different role of each Ca(2+)-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca(2+) entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca(2+) events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca(2+) dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.

Insights into the residence in lipid rafts of adenylyl cyclase AC8 and its regulation by capacitative calcium entry.

Adenylyl cyclases (ACs) are a family of critically important signaling molecules that are regulated by multiple pathways. Adenylyl cyclase 8 (AC8) is a Ca(2+) stimulated isoform that displays a selective regulation by capacitative Ca(2+) entry (CCE), the process whereby the entry of Ca(2+) into cells is triggered by the emptying of intracellular stores. This selectivity was believed to be achieved through the localization of AC8 in lipid raft microdomains, along with components of the CCE apparatus. In the present study, we show that an intact leucine zipper motif is required for the efficient N-linked glycosylation of AC8, and that this N-linked glycosylation is important to target AC8 into lipid rafts. Disruption of the leucine zipper by site-directed mutagenesis results in the elimination of N-glycosylated forms and their exclusion from lipid rafts. Mutants of AC8 that cannot be N-glycosylated are not demonstrably associated with rafts, although they can still be regulated by CCE; however, raft integrity is required for the regulation of these mutants. These findings suggest that raft localized proteins in addition to AC8 are needed to mediate its regulation by CCE.

Separate elements within a single IQ-like motif in adenylyl cyclase type 8 impart ca2+/calmodulin binding and autoinhibition.

The ubiquitous Ca(2+)-sensing protein calmodulin (CaM) fulfills its numerous signaling functions through a wide range of modular binding and activation mechanisms. By activating adenylyl cyclases (ACs) 1 and 8, Ca(2+) acting via calmodulin impacts on the signaling of the other major cellular second messenger cAMP. In possessing two CaM-binding domains, a 1-5-8-14 motif at the N terminus and an IQ-like motif (IQlm) at the C terminus, AC8 offers particularly sophisticated regulatory possibilities. The IQlm has remained unexplored beyond the suggestion that it bound CaM, and the larger C2b region of which it is part was involved in the relief of autoinhibition of AC8. Here we attempt to distinguish the function of individual residues of the IQlm. From a complementary approach of in vitro and cell population AC activity assays, as well as CaM binding, we propose that the IQlm alone, and not the majority of the C2b, imparts CaM binding and autoinhibitory functions. Moreover, this duality of function is spatially separated and depends on amino acid side-chain character. Accordingly, residues critical for CaM binding are positively charged and clustered toward the C terminus, and those essential for the maintenance of autoinhibition are hydrophobic and more N-terminal. Secondary structure prediction of the IQlm supports this separation, with an ideally placed break in the alpha-helical nature of the sequence. We additionally find that the N and C termini of AC8 interact, which is an association specifically abrogated by fully Ca(2+)-bound, but not Ca(2+)-free, CaM. These data support a sophisticated activation mechanism of AC8 by CaM, in which the duality of the IQlm function is critical.

Dynamic regulation, desensitization, and cross-talk in discrete subcellular microdomains during beta2-adrenoceptor and prostanoid receptor cAMP signaling.

Dynamic and localized actions of cAMP are central to the generation of discrete cellular events in response to a range of G(s)-coupled receptor agonists. In the present study we have employed a cyclic nucleotide-gated channel sensor to report acute changes in cAMP in the restricted cellular microdomains adjacent to two different G(s)-coupled receptor pathways, beta(2)-adrenoceptors and prostanoid receptors that are expressed endogenously in HEK293 cells. We probed by either selective small interference RNA-mediated knockdown or dominant negative overexpression the contribution of key signaling components in the rapid attenuation of the local cAMP signaling and subsequent desensitization of each of these G-protein-coupled receptor signaling pathways immediately following receptor activation. Direct measurements of cAMP changes just beneath the plasma membrane of single HEK293 cells reveal novel insights into key regulatory roles provided by protein kinase A-RII, beta-arrestin2, cAMP phosphodiesterase-4D3, and cAMP phosphodiesterase-4D5. We provide new evidence for distinct modes of cAMP down-regulation in these two G(s)-linked pathways and show that these distinct G-protein-coupled receptor signaling systems are subject to unidirectional, heterologous desensitization that allows for limited cross-talk between distinct, dynamically regulated pools of cAMP.

The role of calmodulin recruitment in Ca2+ stimulation of adenylyl cyclase type 8.

Ca2+ stimulation of adenylyl cyclase type 8 (AC8) is mediated by calmodulin (CaM). An earlier study identified two CaM binding sites in AC8; one that was apparently not essential for AC8 activity, located at the N terminus, and a second site that was critical for Ca2+ stimulation, found at the C terminus (Gu, C., and Cooper, D. M. F. (1999) J. Biol. Chem. 274, 8012-8021). This study explores the role of these two CaM binding domains and their interaction in regulating AC8 activity, employing binding and functional studies with mutant CaM and modified AC8 species. We report that the N-terminal CaM binding domain of AC8 has a role in recruiting CaM and that this recruitment is essential to permit stimulation by Ca2+ in vivo. Using Ca2+-insensitive mutants of CaM, we found that partially liganded CaM can bind to AC8, but only fully liganded Ca2+/CaM can stimulate AC8 activity. Moreover, partially liganded CaM inhibited AC8 activity in vivo. The results indicate that CaM pre-associates with the N terminus of AC8, and we suggest that this recruited CaM is used by the C terminus of AC8 to mediate Ca2+ stimulation.

A direct interaction between the N terminus of adenylyl cyclase AC8 and the catalytic subunit of protein phosphatase 2A.

Although protein scaffolding complexes compartmentalize protein kinase A (PKA) and phosphodiesterases to optimize cAMP signaling, adenylyl cyclases, the sources of cAMP, have been implicated in very few direct protein interactions. The N termini of adenylyl cyclases are highly divergent, which hints at isoform-specific interactions. Indeed, the Ca(2+)-sensitive adenylyl cyclase 8 (AC8) contains a Ca(2+)/calmodulin binding site on the N terminus that is essential for stimulation of activity by the capacitative entry of Ca(2+) in the intact cell. Here, we have used the N terminus of AC8 as a bait in a yeast two-hybrid screen of a human embryonic kidney (HEK) 293 cell cDNA library and identified the catalytic subunit of the serine/threonine protein phosphatase 2A (PP2A(C)) as a binding partner. Confirming the highly specific nature of this novel interaction, glutathione-S-transferase fusion proteins containing the full-length N terminus of AC8 affinity precipitated catalytically active PP2A(C) from both HEK293 and mouse forebrain membranes-the latter a normal source of AC8. The scaffolding subunit of PP2A (PP2A(A); 65 kDa) was also precipitated by the N terminus of AC8, indicating that AC8 may occur in a complex with the PP2A core dimer. The interaction between the N terminus of AC8 and PP2A(C) was antagonized by Ca(2+)/calmodulin. However, PP2A(C) and Ca(2+)/calmodulin did not share identical binding specificities in the N terminus of AC8. PKA-mediated phosphorylation did not influence either calmodulin or PP2A(C) association with AC8. In addition, both PP2A(C) and AC8 occurred in lipid rafts. These findings are the first demonstration of an association between adenylyl cyclase and any downstream element of cAMP signaling.

Localized Na+/H+ exchanger 1 expression protects Ca2+-regulated adenylyl cyclases from changes in intracellular pH.

The Ca2+-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca2+ entry (CCE) in nonexcitable cells. The present study investigates whether this Ca2+-dependent modulation of AC activity is further regulated by local pH changes that can arise beneath the plasma membrane as a consequence of cellular activity. Ca2+ stimulation of AC8 expressed in HEK 293 cells and inhibition of endogenous AC6 in C6-2B glioma cells exhibited clear sensitivity to modest pH changes in vitro. Acid pH (pH 7.14) reduced the Ca2+ sensitivity of both ACs, whereas alkaline pH (pH 7.85) enhanced the responsiveness of the enzymes to Ca2+, compared with controls (pH 7.50). Surprisingly, in the intact cell, the response of AC8 and AC6 to CCE was largely unperturbed by similar changes in intracellular pH (pH(i)), imposed using a weak acid (propionate) or weak base (trimethylamine). A range of hypotheses were tested to identify the mechanism(s) that could underlie this lack of pH effect in the intact cell. The pH sensitivity of CCE in HEK 293 cells is likely to dampen the effects of pH(i) on Ca2+-regulated ACs and may partly explain the discrepancy between in vitro and in vivo data. However, we have found that the Na+/H+ exchanger (NHE), NHE1, is functionally active in these cells, and like AC8 (and AC6) it resides in lipid rafts or caveolae, which may create cellular microdomains where pH(i) is tightly regulated. An abundance of NHE1 in these cellular subdomains may generate a privileged environment that protects the Ca2+-sensitive ACs and other caveolar proteins from local acid shifts.

The cytosolic domains of Ca2+-sensitive adenylyl cyclases dictate their targeting to plasma membrane lipid rafts.

Lipid rafts are specialized, cholesterol-rich domains of the plasma membrane that are enriched in certain signaling proteins, including Ca(2+)-sensitive adenylyl cyclases. This restrictive localization plays a key role in the regulation of the Ca(2+)-stimulable AC8 and the Ca(2+)-inhibitable AC6 by capacitative calcium entry. Interestingly, AC7, a Ca(2+)-insensitive AC, is found in the plasma membrane but is excluded from lipid rafts (Smith, K. E., Gu, C., Fagan, K. A., Hu, B., and Cooper, D. M. F. (2002) J. Biol. Chem. 277, 6025-6031). The mechanisms governing the specific membrane targeting of adenylyl cyclase isoforms remain unknown. To address this issue, a series of chimeras were produced between the raft-targeted AC5 and the non-raft-targeted AC7, involving switching of their major domains. The AC5-AC7 chimeras were expressed in HEK 293 cells and lipid rafts were isolated from the bulk plasma membrane by either detergent-based or non-detergent-based fractionation methods. Additionally, confocal imaging was used to investigate the precise cellular targeting of the chimeras. Surprisingly, the two tandem six-transmembrane domains of AC5 were not required for localization to lipid rafts. Rather, AC5 localization depended on the complete cytoplasmic loops (C1 and C2); constructs with mixed domains were either retained in the endoplasmic reticulum or degraded. Similar conclusions are drawn for the lipid raft localization of the Ca(2+)/calmodulin-stimulable AC8; again, the C1 and C2 domains are critical. Thus, protein-protein interactions may be more important than protein-lipid interactions in targeting these calcium-sensitive enzymes to lipid rafts.

Sustained entry of Ca2+ is required to activate Ca2+-calmodulin-dependent phosphodiesterase 1A.

Regulation of adenylyl cyclases (ACs) by Ca2+ requires capacitative Ca2+ entry (CCE) (Cooper, D. M. F. (2003) Biochem. J. 375, 517-529), but whether Ca2+-sensitive phosphodiesterases (PDEs) are similarly discriminating has never been addressed. In the present study, a variety of conditions were devised to manipulate [Ca2+]i so that we could ask whether PDE1 selectively responds to different modes of elevating [Ca2+]i, viz. Ca2+ released from intracellular stores and various modes of Ca2+ entry. In 1321N1 human astrocytoma cells, the endogenous PDE1 (identified as PDE1A by reverse transcriptase-PCR) was largely insensitive to Ca2+ released from carbachol-sensitive stores but was robustly stimulated by a similar rise in [Ca2+]i due to carbachol-induced Ca2+ influx. Gd3+, which effectively blocked thapsigargin-induced CCE and its effect on PDE1A, also inhibited the activation of PDE1A by carbachol-induced Ca2+ entry. However, non-selective ionomycin-mediated Ca2+ entry also activated PDE1A, so that, unlike Ca2+-sensitive ACs, PDE1A cannot discriminate between the different sources of Ca2+ entry. Fractionation of the cells revealed that the Ca2+-calmodulin-stimulated PDE activity was not present at the plasma membrane but was associated with the cytosol and the organellar compartments of the cell. Therefore, the apparent disparity between PDE1A and ACs is likely to be the consequence of their differential subcellular localization. Nevertheless, in a physiological context, where artificial modes of elevating [Ca2+]i are not available, as with ACs, a dependence on CCE would be evident, and it would be the duration of this influx of Ca2+ that would determine how long PDE1A was activated.