Nanako Masada

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 Ca2+. However, whether AKAPs play a role in the control of AC activity by Ca2+ is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca2+-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca2+ 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 Ca2+-stimulated cAMP production.

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 Ca2+-stimulable adenylyl cyclase 8 (AC8). This sensor, and less stringently targeted versions of Epac2-camps, revealed opposite regulation of cAMP synthesis in response to Ca2+ in GH3B6 pituitary cells. Ca2+ 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.

Direct demonstration of discrete Ca2+ microdomains associated with different isoforms of adenylyl cyclase

Ca2+-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca2+ and cAMP that is a crucial feature of cellular homeostasis. Significantly, these ACs are highly selective for capacitative Ca2+ entry (CCE) over other modes of Ca2+ increase. To directly address the possibility that these ACs reside in discrete Ca2+ microdomains, we tethered a Ca2+ sensor, GCaMP2, to the N-terminus of Ca2+-stimulated AC8. GCaMP2-AC8 measurements were compared with global, plasma membrane (PM)-targeted or Ca2+-insensitive AC2-targeted GCaMP2. In intact cells, GCaMP2-AC8 responded rapidly to CCE, but was largely unresponsive to other types of Ca2+ rise. The global GCaMP2, PM-targeted GCaMP2 and GCaMP2-AC2 sensors reported large Ca2+ fluxes during Ca2+ mobilization and non-specific Ca2+ entry, but were less responsive to CCE than GCaMP2-AC8. Our data reveal that different AC isoforms localize to distinct Ca2+-microdomains within the plasma membrane. AC2, which is regulated via protein kinase C, resides in a microdomain that is exposed to a range of widespread Ca2+ signals seen throughout the cytosol. By contrast, a unique Ca2+ microdomain surrounds AC8 that promotes selectivity for Ca2+ signals arising from CCE, and optimizes CCE-mediated cAMP synthesis. This direct demonstration of discrete compartmentalized Ca2+ signals associated with specific signalling proteins provides a remarkable insight into the functional organization of signalling microdomains.

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 Ca2+/calmodulin (CaM), have discrete neuronal functions. Although the Ca2+ sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca2+/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 Ca2+/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca2+/CaM both in vivo and in vitro and explore the different role of each Ca2+-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca2+ entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca2+ events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca2+ 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 Ca2+ stimulated isoform that displays a selective regulation by capacitative Ca2+ entry (CCE), the process whereby the entry of Ca2+ 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.

Highly functionalized terpyridines as competitive inhibitors of AKAP-PKA interactions.

A good fit: Interactions between A-kinase anchoring proteins (AKAPs) and protein kinase A (PKA) play key roles in a plethora of physiologically relevant processes whose dysregulation causes or is associated with diseases such as heart failure. Terpyridines have been developed as α-helix mimetics for the inhibition of such interactions and are the first biologically active, nonpeptidic compounds that block the AKAP binding site of PKA.

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.

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 Ca2+ signaling systems. These ACs, AC1 and AC8, by decoding discrete Ca2+ 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 Ca2+ ions. These two lobes have differing affinities for Ca2+, 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 [Ca2+]i. We first compared by glutathione S-transferase pull-down assays and offline nanoelectrospray ionization mass spectrometry the interaction of CaM and Ca2+-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 Ca2+ 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 Ca2+-CaM activation of AC1 and AC8, which may underpin their different physiological roles.