AC2 Activation Research Articles

Quantitative phosphoproteomic analysis reveals unique cAMP signaling pools emanating from AC2 and AC6 in human airway smooth muscle cells.

Human airway smooth muscle (HASM) is the primary target of ßAR agonists used to control airway hypercontractility in asthma and chronic obstructive pulmonary disease (COPD). ßAR agonists induce the production of cAMP by adenylyl cyclases (ACs), activate PKA and cause bronchodilation. Several other G-protein coupled receptors (GPCR) expressed in human airway smooth muscle cells transduce extracellular signals through cAMP but these receptors elicit different cellular responses. Some G-protein coupled receptors couple to distinct adenylyl cyclases isoforms with different localization, partly explaining this compartmentation, but little is known about the downstream networks that result. We used quantitative phosphoproteomics to define the downstream signaling networks emanating from cAMP produced by two adenylyl cyclases isoforms with contrasting localization in uman airway smooth muscle. After a short stimulus of adenylyl cyclases activity using forskolin, phosphopeptides were analyzed by LC-MS/MS and differences between cells overexpressing AC2 (localized in non-raft membranes) or AC6 (localized in lipid raft membranes) were compared to control human airway smooth muscle. The degree of AC2 and AC6 overexpression was titrated to generate roughly equal forskolin-stimulated cAMP production. 14 Differentially phosphorylated proteins (DPPs) resulted from AC2 activity and 34 differentially phosphorylated proteins resulted from AC6 activity. Analysis of these hits with the STRING protein interaction tool showed that AC2 signaling is more associated with modifications in RNA/DNA binding proteins and microtubule/spindle body proteins while AC6 signaling is associated with proteins regulating autophagy, calcium-calmodulin (Ca2+/CaM) signaling, Rho GTPases and cytoskeletal regulation. One protein, OFD1, was regulated in opposite directions, with serine 899 phosphorylation increased in the AC6 condition 1.5-fold but decreased to 0.46-fold by AC2. In conclusion, quantitative phosphoproteomics is a powerful tool for deciphering the complex signaling networks resulting from discreet signaling events that occur in cAMP compartments. Our data show key differences in the cAMP pools generated from AC2 and AC6 activity and imply that distinct cellular responses are regulated by these two compartments.

The AC2 Protein of a Bipartite Geminivirus Stimulates the Transcription of the BV1 Gene through Abscisic Acid Responsive Promoter Elements.

Geminiviruses possess single-stranded, circular DNA genomes and control the transcription of their late genes, including BV1 of many bipartite begomoviruses, through transcriptional activation by the early expressing AC2 protein. DNA binding by AC2 is not sequence-specific; hence, the specificity of AC2 activation is thought to be conferred by plant transcription factors (TFs) recruited by AC2 in infected cells. However, the exact TFs AC2 recruits are not known for most viruses. Here, we report a systematic examination of the BV1 promoter (PBV1) of the mungbean yellow mosaic virus (MYMV) for conserved promoter motifs. We found that MYMV PBV1 contains three abscisic acid (ABA)-responsive elements (ABREs) within its first 70 nucleotides. Deleting these ABREs, or mutating them all via site-directed mutagenesis, abolished the capacity of PBV1 to respond to AC2-mediated transcriptional activation. Furthermore, ABRE and other related ABA-responsive elements were prevalent in more than a dozen Old World begomoviruses we inspected. Together, these findings suggest that ABA-responsive TFs may be recruited by AC2 to BV1 promoters of these viruses to confer specificity to AC2 activation. These observations are expected to guide the search for the actual TF(s), furthering our understanding of the mechanisms of AC2 action.

Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors

Mammalian adenylate cyclases (ACs) are pseudoheterodimers with dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. We exchanged both membrane anchors of the AC isoform 2 by the quorum-sensing receptor from Vibrio harveyi, CqsS, which has a ligand, Cholera-Autoinducer-1 (CAI-1). In the chimera, AC activity was stimulated by Gsα, CAI-1 had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation. We report that Gsα stimulation of human AC isoforms 2, 3, 5, and 9 expressed in Sf9 cells is inhibited by serum as is AC activity in membranes isolated from rat brain cortex. AC2 activation by forskolin or forskolin/Gsα was similarly inhibited. Obviously, serum contains as yet unidentified factors affecting AC activity. The data establish a linkage in ACs, in which the membrane anchors, as receptors, transduce extracellular signals to the cytosolic catalytic dimer. A mechanistic three state model of AC regulation is presented compatible with all known regulatory inputs into mammalian ACs. The data allow designating the membrane anchors of mammalian ACs as orphan receptors, and establish a new level of AC regulation.

Development of a High-Throughput Screening Paradigm for the Discovery of Small-Molecule Modulators of Adenylyl Cyclase: Identification of an Adenylyl Cyclase 2 Inhibitor

Adenylyl cyclase (AC) isoforms are implicated in several physiologic processes and disease states, but advancements in the therapeutic targeting of AC isoforms have been limited by the lack of potent and isoform-selective small-molecule modulators. The discovery of AC isoform-selective small molecules is expected to facilitate the validation of AC isoforms as therapeutic targets and augment the study of AC isoform function in vivo. Identification of chemical probes for AC2 is particularly important because there are no published genetic deletion studies and few small-molecule modulators. The present report describes the development and implementation of an intact-cell, small-molecule screening approach and subsequent validation paradigm for the discovery of AC2 inhibitors. The NIH clinical collections I and II were screened for inhibitors of AC2 activity using PMA-stimulated cAMP accumulation as a functional readout. Active compounds were subsequently confirmed and validated as direct AC2 inhibitors using orthogonal and counterscreening assays. The screening effort identified SKF-83566 [8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide] as a selective AC2 inhibitor with superior pharmacological properties for selective modulation of AC2 compared with currently available AC inhibitors. The utility of SKF-83566 as a small-molecule probe to study the function of endogenous ACs was demonstrated in C2C12 mouse skeletal muscle cells and human bronchial smooth muscle cells.

Identification and characterization of novel small molecule modulators of adenylyl cyclase

Adenylyl cyclase (AC) isoforms display distinct regulatory and expression patterns, and have specific roles in fundamental physiological processes. These properties have allowed ACs to be considered as therapeutic targets; however, there are few potent and isoform‐selective small molecule modulators of ACs. The objective of these studies was to identify new AC modulators by developing a high throughput screening assay for small molecule inhibitors of AC2. The NIH Clinical Collections (727 compounds) were screened for inhibition of PMA‐stimulated cAMP accumulation in HEK‐AC2 cells. The most active compounds were prioritized by potency for inhibition of AC2 activity, retention of inhibition in an orthogonal cAMP detection format, and in vitro activity against AC isoforms. Actives were also assessed by examining their effect on PMA‐stimulated ERK1/2 phosphorylation to control for inhibition of AC2 via a direct effect on PKC. Compounds were then tested for AC isoform‐selectivity using additional intact cell assays. Three compounds were identified and validated as AC2 inhibitors with unique selectivity patterns against AC1 and AC5. Subsequent studies used cultured bronchial smooth muscle cells to reveal that the most active compound significantly inhibited AC‐regulated interleukin 6 production, a response that is specifically mediated by AC2. Supported by NIH: MH060397.

Differential Effects of AGS3 Expression on D2L Dopamine Receptor-Mediated Adenylyl Cyclase Signaling

Activator of G protein signaling 3 (AGS3) binds Gα(i) subunits in the GDP-bound state, implicating AGS3 as an important regulator of Gα(i)-linked receptor (e.g., D2 dopamine and μ-opioid) signaling. We examined the ability of AGS3 to modulate recombinant adenylyl cyclase (AC) type 1 and 2 signaling in HEK293 cells following both acute and persistent activation of the D(2L) dopamine receptor (D(2L)DR). AGS3 expression modestly enhanced the potency of acute quinpirole-induced D(2L)DR modulation of AC1 or AC2 activity. AGS3 also promoted desensitization of D(2L)DR-mediated inhibition of AC1, whereas desensitization of D(2L)DR-mediated AC2 activation was significantly attenuated. Additionally, AGS3 reduced D(2L)DR-mediated sensitization of AC1 and AC2. These data suggest that AGS3 is involved in altering G protein signaling in a complex fashion that is effector-specific and dependent on the duration of receptor activation.

Identification of new Gβγ interaction sites in adenylyl cyclase 2

The role of Gβγ in adenylyl cyclase (AC) signaling is complicated due to its role as a conditional activator (AC2, AC4 and AC7) and an inhibitor (AC1, AC3 and AC8). AC2 is stimulated by Gα s and if Gβγ is present the stimulation is synergistic. The precise mechanism of this synergistic activation is still not known. In order to further elucidate the role of Gβγ in AC2 activation by Gα s, peptides derived from the C1 domains of AC2 were synthesized and the ability of the various peptides to regulate AC2 function was tested. Our results identify two new Gβγ-binding sites in the AC2 C1 domain, AC2 C1a 339–360 and AC2 C1b 578–602 that are involved with stimulation of AC2 by Gβγ. These two regions are different from the previously described QEHA motif in the C2 domain of AC2. Further, the recently discovered PFAHL motif was confirmed to bind and to be involved with stimulation of AC2 by Gβγ. These functional studies indicate that multiple regions of AC2 are involved in the interaction with Gβγ.

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.

Dexamethasone-Induced Ras Protein 1 Negatively Regulates Protein Kinase C δ: Implications for Adenylyl Cyclase 2 Signaling

We identified dexamethasone-induced Ras protein 1 (Dexras1) as a negative regulator of protein kinase C (PKC) delta, and the consequences of this regulation have been examined for adenylyl cyclase (EC 4.6.1.1) type 2 (AC2) signaling. Dexras1 expression in human embryonic kidney 293 cells completely abolished dopamine D2 receptor-mediated potentiation of AC2 activity, which is consistent with previous reports of its ability to block receptor-mediated Gbetagamma signaling pathways. In addition, Dexras1 significantly reduced phorbol 12-myristate 13-acetate (PMA)-stimulated AC2 activity but did not alter Galpha(s)-mediated cAMP accumulation. Dexras1 seemed to inhibit PMA stimulation of AC2 by interfering with PKCdelta autophosphorylation. This effect was selective for the delta isoform because Dexras1 did not alter autophosphorylation of PKCalpha or PKCepsilon. Dexras1 disruption of PKCdelta autophosphorylation resulted in a significant blockade of PKC kinase activity as measured by [gamma-32P]ATP incorporation using a PKC-specific substrate. Moreover, Dexras1 and PKCdelta coimmunoprecipitated from whole-cell lysates. Dexras1 did not alter the membrane translocation of PKCdelta; however, the ability of Dexras1 to interfere with PKCdelta autophosphorylation was isoprenylation-dependent as determined using the farnesyltransferase inhibitor methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate (FTI-277) and a CAAX box-deficient Dexras1 (C277S) mutant. PMA-stimulated AC2 activity was also not affected by Dexras1 C277S. Taken as a whole, these data suggest that Dexras1 functionally interacts with PKCdelta at the cellular membrane through an isoprenylation-dependent mechanism to negatively regulate PKCdelta activity. Moreover our study suggests that Dexras1 acts to modulate the activation of AC2 in an indirect fashion by inhibiting both Gbetagamma- and PKC-stimulated AC2 activity. The current study provides a novel role for Dexras1 in signal transduction.

G protein ?1?2 subunits purification and their interaction with adenylyl cyclase

A preliminary study on the interaction of G protein (guanine triphosphate binding protein) beta(1)gamma(2) subunits and their coupled components in cell signal transduction was conducted in vitro. The insect cell lines, Sf9 (Spodoptera frugiperda) and H5 (Trichoplusia ni) were used to express the recombinant protein Gbeta(1)gamma(2). The cell membrane containing Gbeta(1)gamma(2) was isolated through affinity chromatography column with Ni-NTAagarose by FPLC method, and the highly purified protein was obtained. The adenylyl cyclase 2 (AC(2)) activity assay showed that the purified Gbeta(1)gamma(2) could significantly stimulate AC(2) activity. The interaction of beta(1)gamma(2) subunits of G protein with the cytoplasmic tail of various mammalian adenylyl cyclases was monitored by BIAcore technology using NTA sensor chip, which relies on the phenomenon of surface plasmon resonance (SPR). The experiments showed the direct binding of Gbeta(1)gamma(2) to the cytoplasmic tail C2 domain of AC2. The specific binding domain of AC2 with Gbeta(1)gamma(2) was the same as AC2 activity domain which was stimulated by Gbeta(1)gamma(2).

The Effect of Nutrient Limitation on Hydrogen Production by Nitrogenase in Continuous Cultures of Azotobacter chroococcum

SUMMARY: H2 production by nitrogenase was investigated in O2-, N2-, C- or SO−2 4-limited continuous cultures of Azotobacter chroococcum. H2 evolution occurred under air and was augmented when Ar replaced N2. Pretreatment of each culture with 40% acetylene in air or Ar/O2 mixtures inhibited the H2-uptake hydrogenase and increased H2 evolution. H2 production in each culture was O2-dependent, and, like acetylene reduction by nitrogenase, it increased to a maximum at an optimum O2 concentration and was inhibited by excess O2. The molar ratio of H2 produced to N2 reduced was least at the optimum O2 concentration and increased sharply under O2-limiting or O2-inhibiting conditions in vivo. The minimum value achieved was approximately 1 in O2-, N2- or C-limited cultures, or with purified nitrogenase components assayed in vitro, and 0·5 in SO4 2−-limited cultures. These differences may reflect different mechanisms of N2 reduction in vivo. Estimates of the levels of nitrogenase component proteins indicated Ac1: Ac2 ratios of 1 or less. However, Ac2 activity may be inhibited under O2-limiting or O2-inhibiting conditions. The maximum ratio of the number of electrons transferred to nitrogenase and to O2 was 0·1 in assays with O2-, C- or SO4 2−-limited cultures; thus, recycling by the H2-uptake hydrogenase of H2 produced by nitrogenase could contribute up to 7% of the total energy produced by respiration.