Adenylyl Cyclase Pathway Research Articles

Functional Selectivity of 9‐OH‐risperidone (paliperidone) versus Risperidone at Dopamine D2 Receptors

Functional selectivity (FS) is the term used to describe the ability of drugs to differentially activate signaling cascades coupled to a single receptor subtype. Importantly, differences in the FS profile between drugs acting at the same receptor subtype may underlie differences in therapeutic efficacy and/or adverse effect liability. Paliperidone (PAL) is the 9‐OH metabolite of risperidone (RIS). Both drugs are effective at treating symptoms of schizophrenia and have similar receptor binding profiles. We compared the FS profiles of PAL and RIS for cellular signaling responses at human D2 receptors expressed in CHO or HEK cells. Relative to the reference ligand butaclamol, the relative efficacy of PAL was greater, not different or less than that of RIS, depending upon the response measured. For example, as inverse agonists at the adenylyl cyclase pathway, the relative efficacy of PAL was greater then RIS. However, for reduction of constitutive desensitization, PAL equaled RIS and for increasing receptor density, the relative efficacy of PAL was less than that of RIS. Thus, a small difference in molecular structure can produce pronounced differences in the FS profile of drugs. Moreover, these results suggest that the presence of 9‐OH group can alter the functional response of RIS at human D2 receptors and suggest that the actions of PAL and RIS in vivo may differ as well.

CRH‐binding protein modulates CRH signaling in a time‐and signaling pathway‐dependent manner

Corticotropin‐releasing hormone (CRH) and the related urocortins (Ucns) mediate the mammalian neuroendocrine stress response by signaling through two G‐protein‐coupled receptors, CRH‐R1 and CRH‐R2. CRH activity at these receptors is also modulated by a secreted CRH‐binding protein (CRH‐BP), which binds CRH/Ucns with equal or greater affinity than the receptors. Initial studies suggested that CRH‐BP predominantly functions to inhibit CRH‐R1 activation, especially at anterior pituitary corticotropes. However, the role of CRH‐BP in CRH signaling via multiple G‐protein pathways has not been fully examined, especially for CRH‐R2. Using purified CRH‐BP and cell culture systems stably‐expressing CRH‐receptors, we determine the modulatory effect of CRH‐BP on CRH‐R1/2 signaling though adenylyl cyclase and MAPK pathways. These studies demonstrate that the role of CRH‐BP is context sensitive, depending on both the specific signaling pathway and whether CRH encounters CRH‐BP prior or simultaneously to the CRH‐receptors. CRH‐receptor activation is inhibited only when CRH is pre‐incubated with CRH‐BP. Without pre‐incubation, CRH‐BP attenuates signal duration but does not prevent receptor activation.This work was supported by NIH T32 GM07544, T32 HD07048, and U. Michigan Biological Chemistry (RTE), NIH DK42730 (AFS), and MDRTC Cell and Molecular Biology Core.

Galanin receptor-1 modulates 5-hydroxtryptamine-1A signaling via heterodimerization

Previous biochemical, cardiovascular and behavioral work has given evidence for the existence of antagonistic galanin receptor-5-HT1A receptor interactions in the brain. In this study we investigated the existence of GalR1-5-HT1A receptor heteromers and their functional characteristics. In mammalian cells transfected with GFP2-tagged 5-HT1A receptor and YFP-tagged GalR1 receptor, a proximity-based fluorescence resonance energy transfer technique was used and it has been demonstrated that GalR1-5-HT1A receptors heteromerize. Furthermore, signaling by either the mitogen-activated protein kinase (MAPK) or adenylyl cyclase (AC) pathways by these heteromers indicates a trans-inhibition phenomenon through their interacting interface via allosteric mechanisms that block the development of an excessive activation of G i/o and an exaggerated inhibition of AC or stimulation of MAPK activity. The presence of these heteromers in the discrete brain regions is postulated based on the existence of GalR-5-HT1A receptor–receptor interactions previously described in the brain and gives rise to explore possible novel therapeutic strategies for treatment of depression by targeting the GalR1-5-HT1A heteromers.

Effect of Hydrogen Sulfide on Cyclic AMP Production in Isolated Bovine and Porcine Neural Retinae

Hydrogen sulfide (H(2)S) has been reported to exert pharmacological effects on neural and non-neural tissues from several mammalian species. In the present study, we examined the role of the intracellular messenger, cyclic AMP in retinal response to H(2)S donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na(2)S) in cows and pigs. Isolated bovine and porcine neural retinae were incubated in oxygenated Krebs buffer solution prior to exposure to varying concentrations of NaHS, Na(2)S or the diterpene activator of adenylate cyclase, forskolin. After incubation at different time intervals, tissue homogenates were prepared for cyclic AMP assay using a well established methodology. In isolated bovine and porcine retinae, the combination of both phosphodiesterase inhibitor, IBMX (2 mM) and forskolin (10 microM) produced a synergistic increase (P < 0.001) in cyclic AMP concentrations over basal levels. NaHS (10 nM-100 microM) produced a time-dependent increase in cyclic AMP concentrations over basal levels which reached a maximum at 20 min in both bovine and porcine retinae. At this time point, both NaHS and Na(2)S (10 nM-100 microM) caused a significant (P < 0.05) dose-dependent increase in cyclic AMP levels in bovine and porcine retinae. For instance, NaHS (100 nM) elicited a four-fold and three-fold increase in cyclic AMP concentrations in bovine and porcine retinae respectively whilst higher concentrations of Na(2)S (100 microM) produced a much lesser effect in both species. In bovine and porcine retinae, the effects caused by forskolin (10 microM) on cyclic AMP production were not potentiated by addition of low or high concentrations of both NaHS and Na(2)S. We conclude that H(2)S donors can increase cyclic AMP production in isolated neural retinae from cows and pigs. Bovine retina appears to be more sensitive to the stimulatory effect of H(2)S donors on cyclic nucleotide production than its porcine counterpart indicating that species differences exist in the magnitude of this response. Furthermore, effects produced by forskolin on cyclic AMP formation were not additive with those elicited by H(2)S donors suggesting that these agents may share a common mechanism in their action on the adenylyl cyclase pathway.

Marked changes in signal transduction upon heteromerization of dopamine D1 and histamine H3 receptors

Functional interactions between the G protein-coupled dopamine D1 and histamine H3 receptors have been described in the brain. In the present study we investigated the existence of D1-H3 receptor heteromers and their biochemical characteristics. D1-H3 receptor heteromerization was studied in mammalian transfected cells with Bioluminescence Resonance Energy Transfer and binding assays. Furthermore, signalling through mitogen-activated protein kinase (MAPK) and adenylyl cyclase pathways was studied in co-transfected cells and compared with cells transfected with either D1 or H3 receptors. Bioluminescence Resonance Energy Transfer and binding assays confirmed that D1 and H3 receptors can heteromerize. Activation of histamine H3 receptors did not lead to signalling towards the MAPK pathway unless dopamine D1 receptors were co-expressed. Also, dopamine D1 receptors, usually coupled to G(s) proteins and leading to increases in cAMP, did not couple to G(s) but to G(i) in co-transfected cells. Furthermore, signalling via each receptor was blocked not only by a selective antagonist but also by an antagonist of the partner receptor. D1-H3 receptor heteromers constitute unique devices that can direct dopaminergic and histaminergic signalling towards the MAPK pathway in a G(s)-independent and G(i)-dependent manner. An antagonist of one of the receptor units in the D1-H3 receptor heteromer can induce conformational changes in the other receptor unit and block specific signals originating in the heteromer. This gives rise to unsuspected therapeutic potentials for G protein-coupled receptor antagonists.

The PDZ Protein Mupp1 Promotes Gi Coupling and Signaling of the Mt1 Melatonin Receptor

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT(1) melatonin receptor and directly regulates its G(i)-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT(1) carboxyl terminus. This high affinity interaction (K(d) approximately 4 nm), which is independent of MT(1) activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT(1)/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT(1), it destabilizes the interaction between MT(1) and G(i) and abolishes G(i)-mediated signaling of MT(1). Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.

Bioluminescence Resonance Energy Transfer Assays Reveal Ligand-specific Conformational Changes within Preformed Signaling Complexes Containing δ-Opioid Receptors and Heterotrimeric G Proteins

Heptahelical receptors communicate extracellular information to the cytosolic compartment by binding an extensive variety of ligands. They do so through conformational changes that propagate to intracellular signaling partners as the receptor switches from a resting to an active conformation. This active state has been classically considered unique and responsible for regulation of all signaling pathways controlled by a receptor. However, recent functional studies have challenged this notion and called for a paradigm where receptors would exist in more than one signaling conformation. This study used bioluminescence resonance energy transfer assays in combination with ligands of different functional profiles to provide in vivo physical evidence of conformational diversity of delta-opioid receptors (DORs). DORs and alpha(i1)beta(1)gamma(2) G protein subunits were tagged with Luc or green fluorescent protein to produce bioluminescence resonance energy transfer pairs that allowed monitoring DOR-G protein interactions from different vantage points. Results showed that DORs and heterotrimeric G proteins formed a constitutive complex that underwent structural reorganization upon ligand binding. Conformational rearrangements could not be explained by a two-state model, supporting the idea that DORs adopt ligand-specific conformations. In addition, conformational diversity encoded by the receptor was conveyed to the interaction among heterotrimeric subunits. The existence of multiple active receptor states has implications for the way we conceive specificity of signal transduction.

Supersensitivity to μ-opioid receptor-mediated inhibition of the adenylyl cyclase pathway involves pertussis toxin-resistant Gα protein subunits

Sustained administration of opioids leads to antinociceptive tolerance, while prolonged association of L-type Ca 2+ channel blockers (e.g. nimodipine) with opioids results in increased antinociceptive response. Herein, we investigated the changes in μ-opioid receptor signalling underlying this shift from analgesic tolerance to supersensitivity. Thus, the interaction of μ-opioid receptors with G proteins and adenylyl cyclase was examined in lumbar spinal cord segments of rats. In control animals, the μ-opioid selective agonists, sufentanil and DAMGO, stimulated [ 35S]5′-(gamma-thio)-triphosphate ([ 35S]GTPγS) binding and inhibited forskolin-stimulated adenylyl cyclase activity, through a mechanism involving pertussis toxin (PTX) sensitive Gα i/o subunits. Seven days of chronic sufentanil treatment developed antinociceptive tolerance associated with a reduction in μ-agonist-induced [ 35S]GTPγS binding, μ-agonist-induced adenylyl cyclase inhibition, and co-precipitation of Gα o, Gα i2 Gα z and Gα q11 subunits with μ-opioid receptors. In contrast, combined nimodipine treatment with sufentanil over the same period increased the sufentanil analgesic response. This antinociceptive supersensitivity was accompanied by a significant increase of μ-agonist-induced inhibition of adenylyl cyclase that was resistant to the antagonism by PTX. In good agreement, co-precipitation of the PTX-resistant, Gα z and Gα q/11 subunits with μ-opioid receptors was not lowered. On the other hand, the PTX-sensitive subunits, Gα i2 and Gα o, as well as agonist-stimulated [ 35S]GTPγS binding were still reduced. Our results demonstrate that μ-opioid analgesic tolerance follows uncoupling of spinal μ-opioid receptors from their G proteins and linked effector pathways. Conversely, the enhanced analgesic response following combined nimodipine treatment with sufentanil is associated with adenylyl cyclase supersensitivity to the opioid inhibitory effect through a mechanism involving PTX-resistant G protein subunits.

Long-term modulation of tyrosine hydroxylase activity and expression by endothelin-1 and -3 in the rat anterior and posterior hypothalamus

Brain catecholamines are involved in the regulation of biological functions, including cardiovascular activity. The hypothalamus presents areas with high density of catecholaminergic neurons and the endothelin system. Two hypothalamic regions intimately related with the cardiovascular control are distinguished: the anterior (AHR) and posterior (PHR) hypothalamus, considered to be sympathoinhibitory and sympathoexcitatory regions, respectively. We previously reported that endothelins (ETs) are involved in the short-term tyrosine hydroxylase (TH) regulation in both the AHR and PHR. TH is crucial for catecholaminergic transmission and is tightly regulated by well-characterized mechanisms. In the present study, we sought to establish the effects and underlying mechanisms of ET-1 and ET-3 on TH long-term modulation. Results showed that in the AHR, ETs decreased TH activity through ET(B) receptor activation coupled to the nitric oxide, phosphoinositide, and CaMK-II pathways. They also reduced total TH level and TH phosphorylated forms (Ser 19 and 40). Conversely, in the PHR, ETs increased TH activity through a G protein-coupled receptor, likely an atypical ET receptor or the ET(C) receptor, which stimulated the phosphoinositide and adenylyl cyclase pathways, as well as CaMK-II. ETs also increased total TH level and the Ser 19, 31, and 40 phosphorylated sites of the enzyme. These findings support that ETs are involved in the long-term regulation of TH activity, leading to reduced sympathoinhibition in the AHR and increased sympathoexcitation in the PHR. Present and previous studies may partially explain the cardiovascular effects produced by ETs when applied to the brain.

Up‐regulation of adenosine A1 receptors in frontal cortex from Pick's disease cases

The adenosine A(1) receptor (A(1)R)-adenylyl cyclase (AC) pathway was studied in post-mortem human frontal and occipital cortex from Pick's disease (PiD) cases and age-matched nondemented controls. In frontal cortex, the main brain area affected in PiD, A(1)Rs, determined by radioligand binding, Western blotting and real-time PCR assays, were significantly increased in PiD samples, suggesting up-regulation of this receptor. AC activity was determined in basal and stimulated conditions via stimulatory guanine nucleotide binding proteins (Gs) using GTP, or directly with forskolin. Basal AC activity was reduced in brains from PiD cases. This agrees with the decrease in AC type I (AC I) level detected by Western blotting. However, inhibition of forskolin-stimulated AC activity by a selective A(1)R agonist was significantly increased in brains from PiD. In occipital cortex, adenosine A(1)R numbers were similar in control and PiD cases, and no significant differences were found in A(1)R-mediated AC inhibition. These results show that the adenosine A(1)R-AC transduction pathway is specifically up-regulated and sensitized in frontal cortex brain in PiD.

Lysophosphatidic Acid Regulates Trafficking of β2-Adrenergic Receptors: THE Gα13/p115RhoGEF/JNK PATHWAY STIMULATES RECEPTOR INTERNALIZATION

Lysophosphatidic acid is an important lipid ligand regulating many aspects of cell function, including proliferation and migration. Operating via heterotrimeric G proteins to downstream effectors, lysophosphatidic acid was shown to regulate the function and trafficking of the G protein-coupled beta(2)-adrenergic receptor. C3 exotoxin, expression of dominant negative RhoA, and inhibition of c-Jun N-terminal kinase blocked the ability of lysophosphatidic acid to sequester the beta(2)-adrenergic receptor, whereas expression of constitutively active Galpha(13), p115RhoGEF, or RhoA mimicked lysophosphatidic acid (LPA) action, stimulating the internalization of the Galpha(s)-coupled beta(2)-adrenergic receptor. This study revealed a novel cross-talk exerted from the LPA/Galpha(13)/p115RhoGEF/RhoA pathway to the beta(2)-adrenergic receptor/Galpha(s)/adenylyl cyclase pathway, attenuating the ability of beta-adrenergic agonists to act following stimulation of cells by LPA as may occur during beta-adrenergic therapy of an inflammatory response.

D1‐like dopamine receptors selectively block P/Q‐type calcium channels to reduce glutamate release onto cholinergic basal forebrain neurones of immature rats

Whole-cell patch-clamp recordings of non-NMDA glutamatergic EPSCs were made from identified cholinergic neurones in slices of basal forebrain (BF) of young rats (P13-P18), to investigate the subtypes of calcium channels involved in dopamine D(1)-like receptor-mediated presynaptic inhibition of the EPSCs. The BF cholinergic neurones were pre-labelled by intracerebroventricular injection of a fluorescent marker, Cy3-192IgG. A D(1)-like receptor agonist, SKF 81297 (30 microM) suppressed the EPSCs reversibly by about 30%, and this inhibition was reproducible. Calcium channel subtypes involved in the glutamatergic transmission were elucidated using selective Ca(2+) channel blockers. The N-type Ca(2+) channel blocker omega-conotoxin (omega-CgTX, 3 microM) suppressed the EPSCs by 57.5%, whereas the P/Q-type channel selective blocker omega-agatoxin-TK (omega-Aga-TK, 200 nM) suppressed the EPSCs by 68.9%. Simultaneous application of both blockers suppressed the EPSCs by 96.1%. The R-type Ca(2+) channel blocker SNX-482 (300 nM) suppressed the EPSCs by 18.4%, whereas nifedipine, the L-type Ca(2+) channel blocker (10 microM), had little effect. In the presence of omega-Aga-TK, SKF 81297, a dopamine D(1)-like receptor agonist, had no effect on the EPSCs. On the other hand, SKF 81297 could still inhibit the EPSCs in the presence of either omega-CgTX, SNX-482 or nifedipine. SKF 81297 had no further effect on the EPSCs when external Ca(2+) concentration was raised to 7.2 mM in the presence of omega-Aga-TK, but could still inhibit the EPSCs in high Ca(2+) solution after omega-CgTX application. Forskolin (FK, 10 microM), an activator of adenylyl cyclase pathway, suppressed the EPSCs, and the FK-induced effect was mostly blocked in the presence of omega-Aga-TK but not that of omega-CgTX. These results suggest that D(1)-like receptor activation selectively blocks P/Q-type calcium channels to reduce glutamate release onto BF cholinergic neurones.

Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay

We developed a functional cell-based high-throughput screening (HTS) assay to identify modulators of the human neuromedin subtype 2 receptor. This assay utilized the signal transduction pathway of hNMU2R, which is positively coupled to adenylyl cyclase and downstream calcium signal pathways. We describe in detail a robust, sensitive, and functional assay for the hNMU2R G-protein-coupled receptor expressed in human embryonic kidney (HEK)-293 cells, whose activity was reflected by a luciferase reporter gene transcriptionally regulated by a 3-repeat serum response element (SRE)-3 repeat multiple response element (MRE)-3 repeat cyclic AMP (cAMP) response element (CRE)-VIP mini promoter. The HEK 293 clonal cell line, stably co-transfected with the 3 × SRE/3 × MRE/3 × CRE/VIP mini promoter-driven luciferase and pCDNA3.1-NMU2R plasmid, was selected by active geneticin sulfate and their ability to express luciferase with a forskolin challenge following hNMU plus forskolin, known to activate intracellular signal transduction. Then the cell density, incubation time, dimethyl sulfoxide (DMSO) concentration used to screen the hNMU receptor subtype 2 specific agonist were optimized, and whether intrinsic luminescent substance of extracts isolated from traditional Chinese herbs disturbs luminescence of luciferase expressed in HEK293 cells was considered. The optimal incubation time was found to be between 8 and 9 h, the cell density and DMSO concentrations were optimized from 3 × 10 4 to 6 × 10 4, and less than 2%, respectively. Our data show that hNMU2R luci-HEK293 cells and their assay exhibit a low background and ideal model for high-throughput screening. These results demonstrate that this reporter gene assay is useful for pharmacological analysis, and is amenable to HTS for human NMU2R agonists.

Gonadotropin-Releasing Hormone Couples to 3′,5′-Cyclic Adenosine-5′-Monophosphate Pathway through Novel Protein Kinase Cδ and -ε in LβT2 Gonadotrope Cells

GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LbetaT2 gonadotrope cells (maximal increase, 2.5-fold; EC(50), 0.30 nm), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca(2+) independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKCdelta, -epsilon, and -theta by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKCdelta or -epsilon but not PKCtheta impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKCdelta and -epsilon are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LbetaT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.

EP2 receptor stimulation promotes calcium responses in astrocytes via activation of the adenylyl cyclase pathway.

Astrocytes are a heterogeneous population of cells that are endowed with a great variety of receptors for neurotransmitters and neuromodulators. Recently prostaglandin E2 has attracted great interest since it is not only released by astrocytes but also activates receptors coupled to either phospholipase C or adenylyl cyclase. We report that EP2 receptor stimulation triggers cAMP production but also causes release of Ca2+ from intracellular stores. This effect is shared by other receptors similarly coupled to adenylyl cyclase and elicited by direct stimulation of the enzyme or application of cAMP analogues. However, the stimulation of the Ca2+ response by cAMP is not mediated by protein kinase A, since a specific antagonist of this kinase had no effect. Such a cross-talk between cAMP and Ca2+ was not observed in all astrocytes. It might therefore reflect a specific resource of either a subpopulation or astrocytes in a specific functional state.

In Vitro and In Vivo Characterization of CK-1827452, a Selective Cardiac Myosin Activator

Current inotropic therapies improve contractility by activating the adenylyl cyclase pathway, by delaying cAMP degradation through inhibition of phosphodiesterase (PDE), or by inhibiting the Na/K exchanger, all of which converge on a final common pathway of increasing the intracellular calcium transient of the cardiac myocyte. However, these calcium dependent treatments can be detrimental to patients with heart failure (HF). A calcium independent approach, increasing cardiac contractility by directly activating cardiac myosin, may address the liabilities of the current inotropic agents. Biochemical specificity, cellular mechanism of action, and in vivo cardiac function in Sprague Dawley (SD) rats, SD rats with HF, and anesthetized dogs was determined with the myosin activator CK-1827452. Biochemical assays demonstrate that CK-1827452 activates the ATPase of an intact cardiac sarcomere by directly activating cardiac myosin. In isolated left ventricular myocytes, CK-1827452 (0.2 μM) increased myocyte contractility without altering the calcium transient. Combination of CK-1827452 with isoproterenol (β-adrenergic agonist) resulted only in an additive increase in contractility with no further change in the calcium transient unlike cAMP dependent inotropic mechanisms. CK-1827452 does not inhibit PDE Type III isolated from human platelets. In vivo contractile function in anesthetized SD rats and dogs was quantitated using echocardiography (M-mode), and simultaneous pressure measurements. CK-1827452 infusion in SD rats (0.25 - 2.5 mg/kg/hr) or dogs (0.03 - 1.0 mg/kg/hr) significantly increased fractional shortening (FS) in a dose-dependent manner with no significant change in peripheral blood pressure or heart rate. Rats with defined heart failure induced by left coronary ligation, or sham treated animals had similar and significant increases in FS when treated with CK-1827452. In summary, CK-1827452 is a cardiac specific myosin activator which increases myocyte contractility without changing the calcium transient, increases FS in rats and dogs, and is efficacious in a rat model of HF. These data indicate that CK-1827452 may be a useful therapeutic in the treatment of human heart failure.

The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy.

Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand-receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O(2))(11)]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (K(d1) = 0.14 +/- 0.02 nM and K(d2) = 1.4 +/- 0.18 nM, and K(d1) = 5.5 +/- 0.7 nM and K(d2) = 620 +/- 117 nM, respectively), whereas only one binding affinity (K(d) = 5.5 +/- 0.4 nM) could be observed for propionyl[Met(O(2))(11)]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).

Gonadotropin regulation of testosterone production by primary cultured theca and granulosa cells of Atlantic croaker: II. Involvement of a mitogen-activated protein kinase pathway

Previous investigations in Atlantic croaker ovaries and primary co-cultured theca and granulosa cells have identified multiple signal transduction pathways involved in the control of gonadotropin-induced steroidogenesis, including adenylyl cyclase- and calcium-dependent signaling pathways. In the present study, evidence was obtained for an involvement of a third signal transduction pathway, a mitogen-activated protein kinase (MAP kinase) signaling cascade, in the regulation of gonadal steroidogenesis in this lower vertebrate teleost model. Gonadotropin-stimulated testosterone synthesis was markedly attenuated by two antagonists of mitogen-activated protein kinase kinases 1/2 (MEK1/2, also known as Map2k1/Map2k2). Moreover, treatment with gonadotropin-induced MEK1/2-dependent phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2, also known as Mapk3/Mapk1) in a concentration- and time-dependent manner in co-cultured croaker theca and granulosa cells. Active MEK1/2 was required for a complete steroidogenic response to activators of the adenylyl cyclase pathway, including forskolin and dbcAMP, suggesting that the target(s) of MAP kinase signaling are distal to cAMP generation and activation of cAMP-dependent protein kinase (PKA). Interestingly, dbcAMP caused a similar increase of ERK1/2 phosphorylation as was observed with gonadotropin treatment, although an inhibitor of PKA did not attenuate this response. Finally, there was no evidence of cross-talk between calcium-dependent signaling pathways and this MAP kinase cascade. While drugs that block calcium-dependent signal transduction, including inhibitors of voltage-sensitive calcium channels, calmodulin, and calcium/calmodulin-dependent kinases, significantly reduced gonadotropin-induced testosterone accumulation, these drugs had no apparent effect on hCG-induced ERK1/2 phosphorylation.

Effect of neurofibromatosis type I mutations on a novel pathway for adenylyl cyclase activation requiring neurofibromin and Ras

Neurofibromatosis type I (NFI) is a common genetic disorder that causes nervous system tumors, and learning and memory defects in humans, and animal models. We identify a novel growth factor stimulated adenylyl cyclase (AC) pathway in the Drosophila brain, which is disrupted by mutations in the epidermal growth factor receptor (EGFR), neurofibromin (NF1) and Ras, but not Galpha(s). This is the first demonstration in a metazoan that a receptor tyrosine kinase (RTK) pathway, acting independently of the heterotrimeric G-protein subunit Galpha(s), can activate AC. We also show that Galpha(s) is the major Galpha isoform in fly brains, and define a second AC pathway stimulated by serotonin and histamine requiring NF1 and Galpha(s), as well as a third, classical Galpha(s)-dependent AC pathway, which is stimulated by Phe-Met-Arg-Phe-amide (FMRFamide) and dopamine. Using mutations and deletions of the human NF1 protein (hNF1) expressed in Nf1 mutant flies, we show that Ras activation by hNF1 is essential for growth factor stimulation of AC activity. Further, we demonstrate that sequences in the C-terminal region of hNF1 are sufficient for NF1/Galpha(s)-dependent neurotransmitter stimulated AC activity, and for rescue of body size defects in Nf1 mutant flies.

Thrombospondin-1 C-terminal-derived peptide protects thyroid cells from ceramide-induced apoptosis through the adenylyl cyclase pathway

Thrombospondin-1, a multi-modular matrix protein is able to interact with a variety of matrix proteins and cell-surface receptors. Thus it is multifunctional. In this work, we examined the role of thrombospondin-1 in ceramide-induced thyroid apoptosis. We focused on the VVM containing sequence localized in the C-terminal domain of the molecule. Primary cultured thyroid cells synthesize thrombospondin-1 depending on their morphological organization. As it leads thyrocytes to organize into monolayers before inducing apoptosis ceramide can modulate this organization. Here, we established that C 2-ceramide treatment decreased thrombospondin-1 expression by interfering with the adenylyl cyclase pathway, thus leading to apoptosis. Furthermore, we demonstrated that the thrombospondin-1-derived peptide 4N1 (RFYVVMWK) abolished ceramide-induced thyroid cell death by preventing intracellular cAMP levels from dropping. Finally, we reported that 4N1-mediated inhibition of ceramide-induced apoptosis was consistently associated with a down-regulation of the caspase-3 processing. Integrin-associated protein receptor (IAP or CD47) was identified as a molecular relay mediating the observed 4N1 effects. Taken together, our results shed light for the first time on anti-apoptotic activities of the thrombospondin-1-derived peptide 4N1 and provide new information on how thrombospondin-1 may control apoptosis of non-tumoral cells.