Sebastian Wachten

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.

Capacitative Ca2+ Entry via Orai1 and Stromal Interacting Molecule 1 (STIM1) Regulates Adenylyl Cyclase Type 8

Capacitative Ca2+ entry (CCE), which occurs through the plasma membrane as a result of Ca2+ store depletion, is mediated by stromal interacting molecule 1 (STIM1), a sensor of intracellular Ca2+ 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 Ca2+ entry reconstituted by coexpression of Orai1 and STIM1 has the functional properties of CCE, we used the Ca2+-calmodulin stimulated adenylyl cyclase type 8 (AC8), which responds selectively to CCE, whereas other modes of Ca2+ entry, including those activated by arachidonate and the ionophore ionomycin, are ineffective. In addition, the Ca2+ 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 Ca2+-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.

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.

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.

Role of phosphodiesterase and adenylate cyclase isozymes in murine colonic glucagon-like peptide 1 secreting cells

Glucagon‐like peptide‐1 (GLP‐1) is secreted from enteroendocrine L‐cells after food intake. Increasing GLP‐1 signalling either through inhibition of the GLP‐1 degrading enzyme dipeptidyl‐peptidase IV or injection of GLP‐1‐mimetics has recently been successfully introduced for the treatment of type 2 diabetes. Boosting secretion from the L‐cell has so far not been exploited, due to our incomplete understanding of L‐cell physiology. Elevation of cyclic adenosine monophosphate (cAMP) has been shown to be a strong stimulus for GLP‐1 secretion and here we investigate the activities of adenylate cyclase (AC) and phosphodiesterase (PDE) isozymes likely to shape cAMP responses in L‐cells.

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.

Muscarinic receptors stimulate AC2 by novel phosphorylation sites, whereas Gβγ 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 Gβγ-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 Gβγ 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 Gβγ subunits, depending on their source.

Separate Elements within a Single IQ-like Motif in Adenylyl Cyclase Type 8 Impart Ca2+/Calmodulin Binding and Autoinhibition

The ubiquitous Ca2+-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, Ca2+ 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 α-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 Ca2+-bound, but not Ca2+-free, CaM. These data support a sophisticated activation mechanism of AC8 by CaM, in which the duality of the IQlm function is critical.