Activation Of cAMP Signaling Research Articles

PDE8 Regulates Rapid Teff Cell Adhesion and Proliferation Independent of ICER

BackgroundAbolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions.Methodology/Principal FindingsWe found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem −/− mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection.Conclusion/SignificanceCollectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.

Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor

The thyrotropin receptor (TSHR) exhibits elevated cAMP signaling in the basal state and becomes fully activated by thyrotropin. Previously we presented evidence that small-molecule ligands act allosterically within the transmembrane region in contrast to the orthosteric extracellular hormone-binding sites. Our goal in this study was to identify positions that surround the allosteric pocket and that are sensitive for inactivation of TSHR. Homology modeling combined with site-directed mutagenesis and functional characterization revealed seven mutants located in the allosteric binding site that led to a decrease of basal cAMP signaling activity. The majority of these silencing mutations, which constrain the TSHR in an inactive conformation, are found in two clusters when mapped onto the 3D structural model. We suggest that the amino acid positions identified herein are indicating locations where small-molecule antagonists, both neutral antagonists and inverse agonists, might interfere with active TSHR conformations.

Convergence of Notch and β-catenin signaling induces arterial fate in vascular progenitors

Molecular mechanisms controlling arterial–venous specification have not been fully elucidated. Previously, we established an embryonic stem cell differentiation system and demonstrated that activation of cAMP signaling together with VEGF induces arterial endothelial cells (ECs) from Flk1+ vascular progenitor cells. Here, we show novel arterial specification machinery regulated by Notch and β-catenin signaling. Notch and GSK3β-mediated β-catenin signaling were activated downstream of cAMP through phosphatidylinositol-3 kinase. Forced activation of Notch and β-catenin with VEGF completely reconstituted cAMP-elicited arterial EC induction, and synergistically enhanced target gene promoter activity in vitro and arterial gene expression during in vivo angiogenesis. A protein complex with RBP-J, the intracellular domain of Notch, and β-catenin was formed on RBP-J binding sites of arterial genes in arterial, but not venous ECs. This molecular machinery for arterial specification leads to an integrated and more comprehensive understanding of vascular signaling.

Signaling networks in somatic cells and oocytes activated during ovulation

The molecular and structural changes in the follicle triggered by the gonadotropin luteinizing hormone (LH) at ovulation require complex interactions between somatic cells and the oocytes. It is well established that the signals originating from the oocytes play an essential role in orchestrating the growth and development of the follicle. Conversely, the exact contribution of local signals in the two compartments to ovulation is less clear. LH causes oocyte meiotic resumption, cumulus expansion, and follicle rupture through direct activation of granulosa cells, but also indirectly by acting on cumulus cells and the oocyte. Recent studies from our and other laboratories have identified some of the components of the signaling network activated at this critical transition. The oocyte meiotic arrest is maintained through the activity of GPCR and adenylyl cyclases endogenous to the oocyte and by diffusion of signals through gap junction connections with the soma. It is possible that cAMP, and more recently, cGMP, diffuses through the gap junction network from somatic cells to the oocyte to maintain meiotic arrest. Recent evidence suggests that the biochemical mechanism by which cGMP maintains meiotic arrest is likely through regulation of the oocyte PDE and therefore, indirectly cAMP. The exact series of events regulating reentry of the oocyte into the cell cycle are still not completely understood, but it is widely accepted that a decrease in cAMP is the primary signal. This decrease in cAMP is caused by an activation of the oocyte PDE but also by closure of the gap junctions. The regulatory mechanisms in the soma that lead to activation of the PDE in the oocytes and gap junction closure are the focus of intense investigation. Several signaling pathways converge on these two final events. LH initially activates cAMP signaling but then the stimulus branches into a number of contiguous and interacting pathways. Activation of the EGF network is one of these pathways and accumulating evidence strongly indicates that this is a component critical for oocyte maturation, cumulus expansion, and follicle rupture. Because these regulatory mechanisms are conserved in rodents and in humans, a better understanding of these signaling networks activated during ovulation will provide new pharmacological opportunities for the manipulation of fertility.

Mutations and polymorphisms in the gene encoding regulatory subunit type 1-alpha of protein kinase A (PRKAR1A): an update

PRKAR1A encodes the regulatory subunit type 1-alpha (RIalpha) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified (online database: http://prkar1a.nichd.nih.gov). The majority are subject to nonsense mediated mRNA decay (NMD), leading to RIalpha haploinsufficiency and, as a result, activated cAMP signaling. Recently, it became apparent that CNC may be caused not only by RIalpha haploinsufficiency, but also by the expression of altered RIalpha protein, as proven by analysis of expressed mutations in the gene, consisting of amino acid substitutions and in-frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered (rather than missing) protein has been analyzed-these are frame-shifts in the 3' end of the coding sequence that shift the stop codon downstream of the normal one. The mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation-negative CNC patients are characterized by significant phenotypic heterogeneity. In this report, we present a comprehensive analysis of all presently known PRKAR1A sequence variations and discuss their molecular context and clinical phenotype.

CAMP/PKA antagonizes thrombin-induced inactivation of endothelial myosin light chain phosphatase: role of CPI-17

Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied. Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells (HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1 complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect. cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent of CPI-17.

Activation of cAMP signaling inhibits DNA damage–induced apoptosis in BCP-ALL cells through abrogation of p53 accumulation

AbstractIn lymphocytes, the second messenger cyclic adenosine monophosphate (cAMP) plays a well-established antiproliferative role through inhibition of G1/S transition and S-phase progression. We have previously demonstrated that, during S-phase arrest, cAMP inhibits the action of S phase–specific cytotoxic compounds, leading to reduction in their apoptotic response. In this report, we provide evidence that cAMP can also inhibit the action of DNA-damaging agents independently of its effect on S phase. Elevation of cAMP in B-cell precursor acute lymphoblastic leukemia cells is shown to profoundly inhibit the apoptotic response to ionizing radiation, anthracyclins, alkylating agents, and platinum compounds. We further demonstrate that this effect depends on the ability of elevated cAMP levels to quench DNA damage–induced p53 accumulation by increasing the p53 turnover, resulting in attenuated Puma and Bax induction, mitochondrial outer membrane depolarization, caspase activation, and poly(ADP-ribose) polymerase cleavage. On the basis of our findings, we suggest that cAMP levels may influence p53 function in malignant cells that retain wild-type p53, potentially affecting p53 both as a tumor suppressor during cancer initiation and maintenance, and as an effector of the apoptotic response to DNA-damaging agents during anticancer treatment.

Chronic Lymphocytic Leukemia and B and T Cells Differ in Their Response to Cyclic Nucleotide Phosphodiesterase Inhibitors

Phosphodiesterase (PDE)4 inhibitors, which activate cAMP signaling by reducing cAMP catabolism, are known to induce apoptosis in B lineage chronic lymphocytic leukemia (CLL) cells but not normal human T cells. The explanation for such differential sensitivity remains unknown. In this study, we report studies contrasting the response to PDE4 inhibitor treatment in CLL cells and normal human T and B cells. Affymetrix gene chip analysis in the three cell populations following treatment with the PDE4 inhibitor rolipram identified a set of up-regulated transcripts with unusually high fold changes in the CLL samples, several of which are likely part of compensatory negative feedback loops. The high fold changes were due to low basal transcript levels in CLL cells, suggesting that cAMP-mediated signaling may be unusually tightly regulated in this cell type. Rolipram treatment augmented cAMP levels and induced ATF-1/CREB serine 63/133 phosphorylation in both B lineage cell types but not T cells. As treatment with the broad-spectrum PDE inhibitor 3-isobutyl-1-methylxanthine induced T cell CREB phosphorylation, we tested a series of family-specific PDE inhibitors for their ability to mimic 3-isobutyl-1-methylxanthine-induced ATF-1/CREB phosphorylation. Whereas PDE3 inhibitors alone had no effect, the combination of PDE3 and PDE4 inhibitors induced ATF-1/CREB serine 63/133 phosphorylation in T cells. Consistent with this observation, PDE3B transcript and protein levels were low in CLL cells but easily detectable in T cells. Combined PDE3/4 inhibition did not induce T cell apoptosis, suggesting that cAMP-mediated signal transduction that leads to robust ATF-1/CREB serine 63/133 phosphorylation is not sufficient to induce apoptosis in this lymphoid lineage.

Protective effect of beraprost sodium, a stable prostacyclin analog, in the development of cigarette smoke extract-induced emphysema

Chronic inflammation, imbalance of proteolytic and anti-proteolytic activities, oxidative stress, and apoptosis of lung structural cells contribute to the pathogenesis of COPD. Prostacyclin protects cells against apoptosis, has anti-inflammatory properties, partially prevents cigarette smoke extract (CSE)-induced apoptosis of the pulmonary endothelium, and thus may be relevant in the pathogenesis of emphysema. We determined whether a synthetic stable prostacyclin analog, beraprost sodium (BPS), attenuates the development of CSE-induced emphysema and elucidated the molecular mechanisms involved in its effect. Sprague-Dawley rats were treated with BPS and injected with CSE once a week for 3 wk. We measured the DNA damage of cells, the expression of caspase-3, and the activity of matrix metalloproteinase (MMP)-2 and MMP-9. We also analyzed TNFalpha and IL-1beta concentrations and the serum antioxidant activity. BPS prevented the development of CSE-induced emphysema, resulting in significant attenuation in alveolar enlargement and pulmonary parenchymal destruction. BPS inhibited pulmonary apoptosis and induction of MMP-2 and MMP-9 activity. Moreover, the protective effect of BPS was associated with a reduction of the expression of proinflammatory cytokines including TNFalpha and IL-1beta and a normalized biological oxidant activity. BPS introduces all these events, probably by activating cAMP signaling through acting specific prostacyclin receptors. In conclusion, BPS protects against the development of CSE-induced emphysema by attenuating apoptosis, inhibiting proteolytic enzyme activity, reducing inflammatory cytokine levels, and augmenting antioxidant activity. BPS may potentially represent a new therapeutic option in the prevention of emphysema in humans in prospect.

Peripheral Olfactory System for Clinical and Basic Psychiatry: A Promising Entry Point to the Mystery of Brain Mechanism and Biomarker Identification in Schizophrenia

Peripheral Olfactory System for Clinical and Basic Psychiatry: A Promising Entry Point to the Mystery of Brain Mechanism and Biomarker Identification in Schizophrenia

Eradication of acute promyelocytic leukemia-initiating cells through PML-RARA degradation

Retinoic acid and arsenic trioxide target the protein stability and transcriptional repression activity of the fusion oncoprotein PML-RARA, resulting in regression of acute promyelocytic leukemia (APL). Phenotypically, retinoic acid induces differentiation of APL cells. Here we show that retinoic acid also triggers growth arrest of leukemia-initiating cells (LICs) ex vivo and their clearance in PML-RARA mouse APL in vivo. Retinoic acid treatment of mouse APLs expressing the fusion protein PLZF-RARA triggers full differentiation, but not LIC loss or disease remission, establishing that differentiation and LIC loss can be uncoupled. Although retinoic acid and arsenic synergize to clear LICs through cooperative PML-RARA degradation, this combination does not enhance differentiation. A cyclic AMP (cAMP)-dependent phosphorylation site in PML-RARA is crucial for retinoic acid-induced PML-RARA degradation and LIC clearance. Moreover, activation of cAMP signaling enhances LIC loss by retinoic acid, identifying cAMP as another potential APL therapy. Thus, whereas transcriptional activation of PML-RARA is likely to control differentiation, its catabolism triggers LIC eradication and long-term remission of mouse APL. Therapy-triggered degradation of oncoproteins could be a general strategy to eradicate cancer stem cells.

Activation of cAMP signaling enhances Fas-mediated apoptosis and activation-induced cell death through potentiation of caspase 8 activation

Defects in Fas receptor signaling lead to compromised maintenance of lymphocyte homeostasis and peripheral immune tolerance, leading in turn to autoimmune disorders. Therefore, agents that can enhance Fas-mediated apoptosis may be therapeutically useful in management of such disorders. In this study, we focused on the effect of cAMP on Fas-mediated apoptosis in human T cells. We show that elevation of intracellular cAMP levels by forskolin, an activator of adenylyl cyclase, 3-isobutyl-1-methylxanthine, an inhibitor of cyclic nucleotide phosphodiesterases, or prostaglandin E 2 potentiates Fas-induced apoptosis in Jurkat cells. Accordingly, cAMP was found to enhance the cleavage of caspase 8 at death-inducing signaling complex and lead to augmentation of the processing of Fas effector proteins. We also demonstrate that cAMP enahnaces Fas-induced apoptosis in normal human T cells and activation-induced cell death in Jurkat cells. These findings provide a rationale for investigating the feasibility of using cAMP-elevating agents to potentiate apoptosis in T cells with aberrant Fas signaling.

Molekulare Mechanismen der Schilddrüsentumorgenese

Schilddrüsenknoten liegen in bis zu 30 % der deutschen Bevölkerung vor und zählen damit zu den häufigsten endokrinologischen Erkrankungen. Schilddrüsenknoten sind außerordentlich heterogen. Die Entstehung von Tumoren mit unterschiedlichen funktionellen und morphologischen Eigenschaften aus einer einzigen Vorläuferzelle ist ein besonderes Charakteristikum der Schilddrüse (SD) und wird durch spezifische genetische Veränderungen verursacht. Bei etwa 60–70 % der differenzierten SD-Karzinome sind Mutationen in bekannten Genen nachweisbar, die über konstitutive Signaltransduktionsaktivierung den Phänotyp des Karzinoms determinieren. In sporadischen papillären SD-Karzinomen (PTCs) liegen BRAF- (oder seltener RAS-)Mutationen vor, während chromosomale Rearrangements (RET, TRK, AKAP9 / BRAF) vorrangig in strahleninduzierten PTCs auftreten. Diese genetischen Veränderungen führen zur MAPKinasen-Aktivierung. In follikulären SD-Karzinomen (FTC) liegen RAS-Mutationen oder PAX8 / PPARγ-Rearrangements vor, diese sind allerdings auch in einem Teil der follikulären Adenome (FA) nachgewiesen. Des Weiteren zeigen aktuelle Arbeiten, dass eine Aktivierung der PI3K / AKT-Signaltransduktion in follikulären SD-Tumoren besonders häufig ist. Undifferenzierte (anaplastische) Schilddrüsenkarzinome (ATC) weisen genetische Merkmale von FTC oder PTC auf und darüber hinaus eine drastische Aktivierung mehrerer Tyrosinkinasekaskaden (Überexpression oder Mutationen im PI3K und MAPKinase-Pathway). Dies unterstreicht das Konzept einer sequenziellen Entwicklung eines ATC aus einem differenzierten SD-Karzinom, als dessen Triggermechanismus die p53-Inaktivierung angesehen wird.

CAMP-dependent signaling as a core component of the mammalian circadian pacemaker.

The mammalian circadian clockwork is modeled as transcriptional and posttranslational feedback loops, whereby circadian genes are periodically suppressed by their protein products. We show that adenosine 3',5'-monophosphate (cAMP) signaling constitutes an additional, bona fide component of the oscillatory network. cAMP signaling is rhythmic and sustains the transcriptional loop of the suprachiasmatic nucleus, determining canonical pacemaker properties of amplitude, phase, and period. This role is general and is evident in peripheral mammalian tissues and cell lines, which reveals an unanticipated point of circadian regulation in mammals qualitatively different from the existing transcriptional feedback model. We propose that daily activation of cAMP signaling, driven by the transcriptional oscillator, in turn sustains progression of transcriptional rhythms. In this way, clock output constitutes an input to subsequent cycles.

Activation of Cyclic AMP Signaling in Ae2-deficient Mouse Fibroblasts

Anion exchanger 2 (AE2, SLC4A2) is a ubiquitously expressed membrane solute carrier that regulates intracellular pH (pH(i)) by exchanging cytosolic bicarbonate for extracellular chloride. We used fibroblasts from Ae2-deficient (Ae2(a,b)(-/-)) mice to study the effects of an alkaline shift in resting intracellular pH (pH(i)) on the activation of cAMP signaling and gene expression. Ae2(a,b)(-/-) fibroblasts show increased pH(i) (by 0.22 +/- 0.03 unit) compared with wild type cells at extracellular pH (pH(o)) 7.4 and 37 degrees C. This shift in resting pH(i) is associated with an up-regulation of bicarbonate-activated soluble adenylyl cyclase expression, increased cAMP production, Creb phosphorylation, inducible cAMP early repressor 1 mRNA expression, and impaired activation of c-Fos transcription by forskolin. These results highlight the importance of bicarbonate transport via Ae2 in maintaining pH(i) homeostasis in cultured mouse fibroblasts and unveil the role of cAMP in the cellular response to chronic alkalization, which putatively includes an inducible cAMP early repressor 1-mediated attenuation of phosphorylated Creb activity.

How Do Bugs Smell?

Two papers published this week report both clarifying and somewhat contradictory findings regarding signaling by insect odorant receptors. Odorant receptors (ORs) in mammals are canonical heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and are often coupled to generation of the second messenger adenosine 3′,5′-monophosphate (cAMP), which can modulate conductance of ion channels. Insect ORs have presented a puzzle, however, as they only vaguely resemble GPCRs and assemble as heterodimers of one variable OR and a common co-receptor called Or83b. Wicher et al . expressed Drosophila receptors Or22a and Or83b in human embryonic kidney cells and used patch clamping to characterize odorant-induced ion currents in the transfected cells. They report that expression of Or83b alone conferred a nonselective cation channel whose conductance was increased by cAMP or guanosine 3′,5′-monophosphate (cGMP). When the authors transfected cells with the Or22a-Or83b pair and also with known mammalian cyclic nucleotide-gated channels, odorant-induced activation of the mammalian channel indicated that the insect OR activated cAMP signaling. Thus, they conclude that the insect ORs themselves form ligand-gated ion channels that sense odorant and amplify signals by G-protein signaling. Sato et al . used similar strategies to analyze various insect ORs. Human cells expressing receptor proteins from fruit fly, mosquito, or silk moth showed a response to odorant that included Ca 2+ influx and increased nonselective cation conductance. Outside-out patch clamp studies from membranes of oocytes or human cells expressing the receptor proteins confirmed that the insect ORs themselves conferred the observed currents. However, Sato et al .’s experiments did not detect odorant-induced generation of cAMP, and pharmacological inhibition of G-protein signaling failed to block odorant-induced currents. As noted in commentary from Chesler and Firestein, resolution of the discrepancies will require further work but may have to do with Sato et al .’s focus on early events (within seconds) and Wicher et al .’s characterization of later events—30 seconds to a minute after receptor stimulation. Insect ORs do not resemble known ion channels at all, so the work suggests there is more to learn about such proteins and raises the question of whether orphan GPCRs in other organisms might also function as ligand-gated ion channels. K. Sato, M. Pellegrino, T. Nakagawa, T. Nakagawa, L. B. Vosshall, K. Touhara, Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature 452 , 1002-1006 (2008). [PubMed] D. Wicher, R. Schäfer, R. Bauernfeind, M. C. Stensmyr, R. Heller, S. H. Heinemann, B. S. Hannson, Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452 , 1007-1011 (2008). [PubMed] A. Chesler, S. Firestein, Current views on odour receptors. Nature 452 , 944 (2008). [PubMed]

Cyclic AMP-mediated growth suppression and MAPK phosphorylation in thyroid papillary carcinoma cells

The main regulating systems of thyroid growth are the mitogen-activated protein kinase (MAPK) signaling pathway and the cAMP signaling pathway. Thyroid papillary carcinoma frequently involves mutations in BRAF or RET/PTC without overlap, which are expected to constitutively activate MAPK signaling. On the other hand, it has been reported that cAMP signaling acts in an inhibitory manner on the proliferation of papillary carcinoma cell lines, although the cAMP pathway physiologically promotes the proliferation of normal follicular cells as well as hormonogenesis. The effect of cAMP on proliferation is attributed to crosstalk with MAPK signaling. However, this phenomenon has not been clearly established in papillary carcinoma with BRAF or RET/PTC mutations. In order to elucidate whether activated cAMP signaling inhibits cell proliferation and affects MAPK signaling in papillary carcinoma, we performed in vitro experiments using two representative cell lines, K1 and TPC-1, which have a BRAF and an RET/PTC mutation, respectively. Elevated cAMP caused by an adenylate cyclase activator suppressed the proliferation of both K1 and TPC-1 cells. Examining the crosstalk between cAMP and MAPK signaling, K1 and TPC-1 cells showed opposite responses to cAMP activation. These responses were blocked by an inhibitor of the cAMP-dependent protein kinase (PKA). In K1 cells, B-Raf might predominate over Raf-1, and the elevated cAMP is thought to promote MAPK phosphorylation through the PKA-mediated activation of Rap1. On the other hand, in TPC-1 cells Raf-1 might predominate and could be inhibited by activated Rap1, resulting in the suppression of MAPK phosphorylation. In conclusion, the proliferation of both papillary carcinoma cell types was significantly suppressed by cAMP signaling, regardless of whether MAPK signaling was activated or inactivated by the PKA-mediated cAMP signaling pathway. There could, however, be other mechanisms by which cAMP signaling inhibits the growth of papillary carcinoma cells.

Expression, binding, and signaling properties of CRF2(a)receptors endogenously expressed in human retinoblastoma Y79 cells: passage‐dependent regulation of functional receptors

Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF(1)-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of approximately 100 pmoles/10(5) cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of approximately 25-30 pmoles/10(5) cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the approximately 300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 +/- 1.6 nmol/L), whereas the CRF(1)-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 microL. When the CRF(1)-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 +/- 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 +/- 0.2 microL). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2a receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture.

The human vomeronasal type‐1 receptor family—detection of volatiles and cAMP signaling in HeLa/Olf cells

The human genome harbors 5 remnant genes coding for vomeronasal type-1 receptors, compared with 187 of such receptors in mice. In rodents, vomeronasal type-1 receptors are typically expressed in the vomeronasal organ. They are believed to be highly selective and sensitive pheromone detectors, as may be inferred from one receptor, V1rb2, responding to picomolar concentrations of the mouse pheromone 2-heptanone. Expression patterns, ligands, and signal transduction of human vomeronasal type-1 receptors have, however, remained largely obscure. Our aim was to deorphan and functionally characterize these 5 putative human pheromone receptors. Here, we report functional expression for all 5 receptors in HeLa/Olf cells. The recombinant, N-terminally tagged receptors expressed at the plasma membrane of HeLa/Olf cells and responded differentially to 19 of 140 odorants in a combinatorial way. C9-C10 aliphatic alcohols or aldehydes emerged as the best agonists at submicromolar concentrations above human odorant thresholds. Surprisingly, and in contrast to mouse V1rb2, all human vomeronasal type-1 receptors activated cAMP signaling via G protein alphaolf, when expressed in HeLa/Olf cells. While a biological function of human vomeronasal type-1 receptors is still elusive, our data show that their major functional characteristics are similar to those of odorant receptors.

Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP

cAMP is well known to regulate exocytosis in various secretory cells, but the precise mechanism of its action remains unknown. Here, we examine the role of cAMP signaling in the exocytotic process of insulin granules in pancreatic beta cells. Although activation of cAMP signaling alone does not cause fusion of the granules to the plasma membrane, it clearly potentiates both the first phase (a prompt, marked, and transient increase) and the second phase (a moderate and sustained increase) of glucose-induced fusion events. Interestingly, all granules responsible for this potentiation are newly recruited and immediately fused to the plasma membrane without docking (restless newcomer). Importantly, cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis are markedly reduced in mice lacking the cAMP-binding protein Epac2 (Epac2(ko/ko)). In addition, the small GTPase Rap1, which is activated by cAMP specifically through Epac2 in pancreatic beta cells, mediates cAMP-induced insulin secretion in a protein kinase A-independent manner. We also have developed a simulation model of insulin granule movement in which potentiation of the first phase is associated with an increase in the insulin granule density near the plasma membrane. Taken together, these data indicate that Epac2/Rap1 signaling is essential in regulation of insulin granule dynamics by cAMP, most likely by controlling granule density near the plasma membrane.