Mycobacterium tuberculosis PhoP integrates stress response to intracellular survival by regulating cAMP level.

Survival of Mycobacterium tuberculosis within the host macrophages requires the bacterial virulence regulator PhoP, but the underlying reason remains unknown. 3',5'-Cyclic adenosine monophosphate (cAMP) is one of the most widely used second messengers, which impacts a wide range of cellular responses in microbial pathogens including M. tuberculosis. Herein, we hypothesized that intra-bacterial cAMP level could be controlled by PhoP since this major regulator plays a key role in bacterial responses against numerous stress conditions. A transcriptomic analysis reveals that PhoP functions as a repressor of cAMP-specific phosphodiesterase (PDE) Rv0805, which hydrolyzes cAMP. In keeping with these results, we find specific recruitment of the regulator within the promoter region of rv0805 PDE, and absence of phoP or ectopic expression of rv0805 independently accounts for elevated PDE synthesis, leading to the depletion of intra-bacterial cAMP level. Thus, genetic manipulation to inactivate PhoP-rv0805-cAMP pathway decreases cAMP level, stress tolerance, and intracellular survival of the bacillus.

The cAMP-dependent phosphorylation footprint in response to heat stress.

KEY MESSAGE: cAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress. In plants, 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP-dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular "sponge" that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

Features of Intracellular Signal Transduction in Neural Stem Cells under the Influence of Alkaloid Songorine, an Agonist of Fibroblast Growth Factor Receptors.

We performed a comparative in vitro study of the involvement of NF-κB, PI3K, cAMP, ERK1/2, p38, JAKs, STAT3, JNK, and p53-dependent intracellular signaling in the functioning of neural stem cells (NSC) under the influence of basic fibroblast growth factor (FGF) and FGF receptor agonist, diterpene alkaloid songorine. The significant differences in FGFR-mediated intracellular signaling in NSC were revealed for these ligands. In both cases, stimulation of progenitor cell proliferation occurs with the participation of NF-κB, PI3K, ERK1/2, JAKs, and STAT3, while JNK and p53, on the contrary, inhibit cell cycle progression. However, under the influence of songorin, cAMP- and p38-mediated cascades are additionally involved in the transmission of the NSC division-activating signal. In addition, unlike FGF, the alkaloid stimulates progenitor cell differentiation by activating ERK1/2, p38, JNK, p53, and STAT3.

Presynaptic structural and functional plasticity are coupled by convergent Rap1 signaling.

Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of Rut-cAMP-Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton.

Therapeutic potentials of nonpeptidic V2R agonists for partial cNDI-causing V2R mutants.

Loss-of-function mutations in the type 2 vasopressin receptor (V2R) are a major cause of congenital nephrogenic diabetes insipidus (cNDI). In the context of partial cNDI, the response to desmopressin (dDAVP) is partially, but not entirely, diminished. For those with the partial cNDI, restoration of V2R function would offer a prospective therapeutic approach. In this study, we revealed that OPC-51803 (OPC5) and its structurally related V2R agonists could functionally restore V2R mutants causing partial cNDI by inducing prolonged signal activation. The OPC5-related agonists exhibited functional selectivity by inducing signaling through the Gs-cAMP pathway while not recruiting ß-arrestin1/2. We found that six cNDI-related V2R partial mutants (V882.53M, Y1283.41S, L1614.47P, T2736.37M, S3298.47R and S3338.51del) displayed varying degrees of plasma membrane expression levels and exhibited moderately impaired signaling function. Several OPC5-related agonists induced higher cAMP responses than AVP at V2R mutants after prolonged agonist stimulation, suggesting their potential effectiveness in compensating impaired V2R-mediated function. Furthermore, docking analysis revealed that the differential interaction of agonists with L3127.40 caused altered coordination of TM7, potentially contributing to the functional selectivity of signaling. These findings suggest that nonpeptide V2R agonists could hold promise as potential drug candidates for addressing partial cNDI.

Sensing antibody functions with a novel CCR8-responsive engineered cell.

Human chemokine receptor 8 (CCR8) is a promising drug target for immunotherapy of cancer and autoimmune diseases. Monoclonal antibody-based CCR8 targeted treatment shows significant inhibition in tumor growth. The inhibition of CCR8 results in the improvement of antitumor immunity and patient survival rates by regulating tumor-resident regulatory T cells. Recently monoclonal antibody drug development targeting CCR8 has become a research hotspot, which also promotes the advancement of antibody evaluation methods. Therefore, we constructed a novel engineered customized cell line HEK293-cAMP-biosensor-CCR8 combined with CCR8 and a cAMP-biosensor reporter. It can be used for the detection of anti-CCR8 antibody functions like specificity and biological activity, in addition to the detection of antibody-dependent cell-mediated cytotoxicity and antibody-dependent-cellular-phagocytosis. We obtained a new CCR8 mAb 22H9 and successfully verified its biological activities with HEK293-cAMP-biosensor-CCR8. Our reporter cell line has high sensitivity and specificity, and also offers a rapid kinetic detection platform for evaluating anti-CCR8 antibody functions.

Synergistic induction of blood-brain barrier properties.

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell-derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/ß-catenin signaling and inhibition of the TGF-ß pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/ß-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.

Forskolin-mediated cAMP activation upregulates TNF-α expression despite NF-κB downregulation in LPS-treated Schwann cells.

Although Schwann cells have been found to play a key role in inflammation and repair following nerve injury, the exact pathway is still unknown. To explore the mechanism by which Schwann cells exert their effects in the neuron microenvironment, we investigated two main inflammatory pathways: the NF-κB and cAMP pathways, and their downstream signaling molecules. In this study, lipopolysaccharide (LPS), a bacterial endotoxin, was used to activate the NF-κB pathway, and forskolin, a plant extract, was used to activate the cAMP pathway. The rat RT4-D6P2T Schwann cell line was treated with 0.1, 1, or 10 µg/mL of LPS, with or without 2 µM of forskolin, for 1, 3, 12, and 24 hours to determine the effects of elevated cAMP levels on LPS-treated cell viability. To investigate the effects of elevated cAMP levels on the expression of downstream signaling effector proteins, specifically NF-κB, TNF-α, AKAP95, and cyclin D3, as well as TNF-α secretion, RT4-D6P2T cells were incubated in the various treatment combinations for a 3-hour time period. Overall, results from the CellTiter-Glo viability assay revealed that forskolin increased viability in cells treated with smaller doses of LPS for 1 and 24 hours. For all time points, 10 µg/mL of LPS noticeably reduced viability regardless of forskolin treatment. Results from the Western blot analysis revealed that, at 10 µg/mL of LPS, forskolin upregulated the expression of TNF-α despite a downregulation of NF-κB, which was also accompanied by a decrease in TNF-α secretion. These results provide evidence that cAMP might regulate TNF-α expression through alternate pathways. Furthermore, although cAMP activation altered AKAP95 and cyclin D3 expression at different doses of LPS, there does not appear to be an association between the expression of AKAP95 or cyclin D3 and the expression of TNF-α. Exploring the possible interactions between cAMP, NF-κB, and other key inflammatory signaling pathways might reveal a potential therapeutic target for the treatment of nerve injury and inflammation.

Targeting phosphodiesterase 4 as a potential therapy for Parkinson's disease: a review.

Phosphodiesterases (PDEs) have become a promising therapeutic target for various disorders. PDEs are a vast and diversified family of enzymes that degrade cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have several biochemical and physiological functions. Phosphodiesterase 4 (PDE4) is the most abundant PDE in the central nervous system (CNS) and is extensively expressed in the mammalian brain, where it catalyzes the hydrolysis of intracellular cAMP. An alteration in the balance of PDE4 and cAMP results in the dysregulation of different biological mechanisms involved in neurodegenerative diseases. By inhibiting PDE4 with drugs, the levels of cAMP inside the cells could be stabilized, which may improve the symptoms of mental and neurological disorders such as memory loss, depression, and Parkinson's disease (PD). Though numerous studies have shown that phosphodiesterase 4 inhibitors (PDE4Is) are beneficial in PD, there are presently no approved PDE4I drugs for PD. This review presents an overview of PDE4Is and their effects on PD, their possible underlying mechanism in the restoration/protection of dopaminergic cell death, which holds promise for developing PDE4Is as a treatment strategy for PD. Methods on how these drugs could be effectively delivered to develop as a promising treatment for PD have been suggested.

Urocortin 3 contributes to paracrine inhibition of islet alpha cells in mice.

Pancreatic alpha cell activity and glucagon secretion lower as glucose levels increase. While part of the decrease is regulated by glucose itself, paracrine signaling by their neighboring beta and delta cells also plays an important role. Somatostatin from delta cells is an important local inhibitor of alpha cells at high glucose. Additionally, urocortin 3 (UCN3) is a hormone that is co-released from beta cells with insulin and acts locally to potentiate somatostatin secretion from delta cells. UCN3 thus inhibits insulin secretion via a negative feedback loop with delta cells, but its role with respect to alpha cells and glucagon secretion is not understood. We hypothesize that the somatostatin-driven glucagon inhibition at high glucose is regulated in part by UCN3 from beta cells. Here, we use a combination of live functional Ca2+ and cAMP imaging as well as direct glucagon secretion measurement, all from alpha cells in intact mouse islets, to determine the contributions of UCN3 to alpha cell behavior. Exogenous UCN3 treatment decreased alpha cell Ca2+ and cAMP levels and inhibited glucagon release. Blocking endogenous UCN3 signaling increased alpha cell Ca2+ by 26.8 ± 7.6%, but this did not result in increased glucagon release at high glucose. Furthermore, constitutive deletion of Ucn3 did not increase Ca2+ activity or glucagon secretion relative to controls. UCN3 is thus capable of inhibiting mouse alpha cells, but, given the subtle effects of endogenous UCN3 signaling on alpha cells, we propose that UCN3-driven somatostatin may serve to regulate local paracrine glucagon levels in the islet instead of inhibiting gross systemic glucagon release.

The cAMP signaling module regulates sperm motility in the liverwort Marchantia polymorpha.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a universal signaling molecule that acts as a second messenger in various organisms. It is well established that cAMP plays essential roles across the tree of life, although the function of cAMP in land plants has long been debated. We previously identified the enzyme with both adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) activity as the cAMP-synthesis/hydrolysis enzyme COMBINED AC with PDE (CAPE) in the liverwort Marchantia polymorpha. CAPE is conserved in streptophytes that reproduce with motile sperm; however, the precise function of CAPE is not yet known. In this study, we demonstrate that the loss of function of CAPE in M. polymorpha led to male infertility due to impaired sperm flagellar motility. We also found that two genes encoding the regulatory subunits of cAMP-dependent protein kinase (PKA-R) were also involved in sperm motility. Based on these findings, it is evident that CAPE and PKA-Rs act as a cAMP signaling module that regulates sperm motility in M. polymorpha. Therefore, our results have shed light on the function of cAMP signaling and sperm motility regulators in land plants. This study suggests that cAMP signaling plays a common role in plant and animal sperm motility.

A cAMP-biosensor-based assay for measuring plasma arginine-vasopressin levels.

Arginine-vasopressin (AVP), a cyclic peptide hormone composed of nine amino acids, regulates water reabsorption by increasing intracellular cyclic adenosine monophosphate (cAMP) concentrations via the vasopressin V2 receptor (V2R). Plasma AVP is a valuable biomarker for the diagnosis of central diabetes insipidus (CDI) and is commonly measured using radioimmunoassay (RIA). However, RIA has several drawbacks, including a long hands-on time, complex procedures, and handling of radioisotopes with special equipment and facilities. In this study, we developed a bioassay to measure plasma AVP levels using HEK293 cells expressing an engineered V2R and a cAMP biosensor. To achieve high sensitivity, we screened V2R orthologs from 11 various mammalian species and found that the platypus V2R (pV2R) responded to AVP with approximately six-fold higher sensitivity than that observed by the human V2R. Furthermore, to reduce cross-reactivity with desmopressin (DDAVP), a V2R agonist used for CDI treatment, we introduced a previously described point mutation into pV2R, yielding an approximately 20-fold reduction of responsiveness to DDAVP while maintaining responsiveness to AVP. Finally, a comparison of plasma samples from 12 healthy individuals demonstrated a strong correlation (Pearson's correlation value: 0.90) between our bioassay and RIA. Overall, our assay offers a more rapid and convenient method for quantifying plasma AVP concentrations than existing techniques.

Pharmacological potential of cyclic nucleotide signaling in immunity.

Cyclic nucleotides are important signaling molecules that play many critical physiological roles including controlling cell fate and development, regulation of metabolic processes, and responding to changes in the environment. Cyclic nucleotides are also pivotal regulators in immune signaling, orchestrating intricate processes that maintain homeostasis and defend against pathogenic threats. This review provides a comprehensive examination of the pharmacological potential of cyclic nucleotide signaling pathways within the realm of immunity. Beginning with an overview of the fundamental roles of cAMP and cGMP as ubiquitous second messengers, this review delves into the complexities of their involvement in immune responses. Special attention is given to the challenges associated with modulating these signaling pathways for therapeutic purposes, emphasizing the necessity for achieving cell-type specificity to avert unintended consequences. A major focus of the review is on the recent paradigm-shifting discoveries regarding specialized cyclic nucleotide signals in the innate immune system, notably the cGAS-STING pathway. The significance of cyclic dinucleotides, exemplified by 2'3'-cGAMP, in controlling immune responses against pathogens and cancer, is explored. The evolutionarily conserved nature of cyclic dinucleotides as antiviral agents, spanning across diverse organisms, underscores their potential as targets for innovative immunotherapies. Findings from the last several years have revealed a striking diversity of novel bacterial cyclic nucleotide second messengers which are involved in antiviral responses. Knowledge of the existence and precise identity of these molecules coupled with accurate descriptions of their associated immune defense pathways will be essential to the future development of novel antibacterial therapeutic strategies. The insights presented herein may help researchers navigate the evolving landscape of immunopharmacology as it pertains to cyclic nucleotides and point toward new avenues or lines of thinking about development of therapeutics against the pathways they regulate.

miR-145a-5p/SIK1/cAMP-dependent alteration of synaptic structural plasticity drives cognitive impairment induced by coke oven emissions.

Exposure to fine particulate matter (PM) is associated with the neurodegenerative diseases. Coke oven emissions (COEs) in occupational environment are important sources of PM. However, its neurotoxicity is still unclear. Therefore, evaluating the toxicological effects of COE on the nervous system is necessary. In the present study, we constructed mouse models of COE exposure by tracheal instillation. Mice exposed to COE showed signs of cognitive impairment. This was accompanied by a decrease in miR-145a-5p and an increase in SIK1 expression in the hippocampus, along with synaptic structural damage. Our results demonstrated that COE-induced miR-145a-5p downregulation could increase the expression of SIK1 and phosphorylated SIK1, inhibiting the cAMP/PKA/CREB pathway by activating PDE4D, which was associated with reduced synaptic structural plasticity. Furthermore, restoring of miR-145a-5p expression based on COE exposure in HT22 cells could partially reversed the negative effects of COE exposure through the SIK1/PDE4D/cAMP axis. Collectively, our findings link epigenetic regulation with COE-induced neurotoxicity and imply that miR-145a-5p could be an early diagnostic marker for neurological diseases in patients with COE occupational exposure.

The role of soluble adenylyl cyclase in sensing and regulating intracellular pH.

Soluble adenylyl cyclase (sAC) differs from transmembrane adenylyl cyclases (tmAC) in many aspects. In particular, the activity of sAC is not regulated by G-proteins but by the prevailing bicarbonate concentrations inside cells. Therefore, sAC serves as an exquisite intracellular pH sensor, with the capacity to translate pH changes into the regulation of localization and/or activity of cellular proteins involved in pH homeostasis. In this review, we provide an overview of literature describing the regulation of sAC activity by bicarbonate, pinpointing the importance of compartmentalization of intracellular cAMP signaling cascades. In addition, examples of processes involving proton and bicarbonate transport in different cell types, in which sAC plays an important regulatory role, were described in detail.

Maximum entropy determination of mammalian proteome dynamics.

Full understanding of proteostasis and energy utilization in cells will require knowledge of the fraction of cell proteins being degraded with different half-lives and their rates of synthesis. We therefore developed a method to determine such information that combines mathematical analysis of protein degradation kinetics obtained in pulse-chase experiments with Bayesian data fitting using the maximum entropy principle. This approach will enable rapid analyses of whole-cell protein dynamics in different cell types, physiological states, and neurodegenerative disease. Using it, we obtained surprising insights about protein stabilities in cultured cells normally and upon activation of proteolysis by mTOR inhibition and increasing cAMP or cGMP. It revealed that >90% of protein content in dividing mammalian cell lines is long-lived, with half-lives of 24 to 200 h, and therefore comprises much of the proteins in daughter cells. The well-studied short-lived proteins (half-lives < 10 h) together comprise <2% of cell protein mass, but surprisingly account for 10 to 20% of measurable newly synthesized protein mass. Evolution thus appears to have minimized intracellular proteolysis except to rapidly eliminate misfolded and regulatory proteins.

Constitutive internalisation of EP2 differentially regulates G protein signalling.

The prostanoid G protein-coupled receptor (GPCR) EP2 is widely expressed and implicated in endometriosis, osteoporosis, obesity, pre-term labour and cancer. Internalisation and intracellular trafficking are critical for shaping GPCR activity, yet little is known regarding the spatial programming of EP2 signalling and whether this can be exploited pharmacologically. Using three EP2-selective ligands that favour activation of different EP2 pathways, we show that EP2 undergoes limited agonist-driven internalisation but is constitutively internalised via dynamin-dependent, ß-arrestin-independent pathways. EP2 was constitutively trafficked to early and very early endosomes (VEE), which was not altered by ligand activation. APPL1, a key adaptor and regulatory protein of the VEE, did not impact EP2 agonist-mediated cAMP. Internalisation was required for ~70% of the acute butaprost- and AH13205-mediated cAMP signalling, yet PGN9856i, a Gαs-biased agonist, was less dependent on receptor internalisation for its cAMP signalling, particularly in human term pregnant myometrial cells that endogenously express EP2. Inhibition of EP2 internalisation partially reduced calcium signalling activated by butaprost or AH13205 and had no effect on PGE2 secretion. This indicates an agonist-dependent differential spatial requirement for Gαs and Gαq/11 signalling and a role for plasma membrane-initiated Gαq/11-Ca2+-mediated PGE2 secretion. These findings reveal a key role for EP2 constitutive internalisation in its signalling and potential spatial bias in mediating its downstream functions. This, in turn, could highlight important considerations for future selective targeting of EP2 signalling pathways.

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ.

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 µM forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

Variable CGRP family peptide signaling durations and the structural determinants thereof.

Calcitonin gene-related peptides alpha and beta (αCGRP, ßCGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) function in pain signaling, neuroimmune communication, and regulation of the cardiovascular and lymphatic systems by activating either of two class B GPCRs, CLR and CTR, in complex with a RAMP1, -2, or -3 modulatory subunit. Inspired by our recent discovery that AM2/IMD(1-47) activation of CLR-RAMP3 elicits long duration cAMP signaling, here we used a live-cell cAMP biosensor assay to characterize the signaling kinetics of the two CGRP peptides and several bioactive AM and AM2/IMD fragments with variable N-terminal extensions. Remarkably, AM2/IMD(8-47) and AM2/IMD-53 exhibited even longer duration signaling than the 1-47 fragment. AM2/IMD(8-47) was a striking 8-fold longer acting than AM(13-52) at CLR-RAMP3. In contrast, the N-terminal extension of AM had no effect on signaling duration. AM(1-52) and (13-52) were equally short-acting. Analysis of AM2/IMD-AM mid-region chimeras and AM2/IMD R23 and R33 point mutants showed the importance of these residues for long-duration signaling and identified AM2/IMD peptides that exhibited up to 17-fold diminished signaling duration at CLR-RAMP3, while retaining near wildtype signaling potencies. ßCGRP was âˆ¼ 3-fold longer acting than αCGRP at the CGRP (CLR-RAMP1) and the amylin1 (CTR-RAMP1) receptors. Chimeric CGRP peptides showed that the single residue difference near the N-terminus, and the two differences in the mid-region, equally contributed to the longer duration of ßCGRP signaling. This work uncovers key temporal differences in cAMP signaling among the CGRP family peptides, elucidates the structural bases thereof, and provides pharmacological tools for studying long-duration AM2/IMD signaling.

Live-cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeast Schizosaccharomyces pombe.

The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.