Guanyl Nucleotide-binding Protein Research Articles

Exploring Light and Life

Exploring Light and Life

Abstract LB-16: Inhibition of RalA as a novel strategy for targeting ovarian cancer

Abstract Abstract: The Ral (Ras like) GTPase guanyl nucleotide-binding proteins, RalA and RalB are direct effectors of pro-oncogene Ras. RalA and RalB are implicated in tumorigenesis and play distinct roles in mediating carcinogenesis. The critical role of RalA in the initiation and progression of ovarian cancer is entirely unclear. Here, we report that RalA is overactivated in human ovarian cancer cells and human ovarian cancer tissues. The RalA downstream effectors (RalBP-1 and CDC42) and the regulators (GalGDS, PP2A Aα and β, as well as Aurora Kinase) that modulate RalA activation during the post-translational process were found to be de-regulated in ovarian cancer cells. ShRNA-mediated knockdown of RalA inhibited ovarian cancer cell proliferation and invasion. Geranyl-Geranyl transferase inhibitors (GGTI-2147) or Aurora Kinase Inhibitor (AKI) that inhibit RalA activation at post-translational levels suppressed ovarian cancer cell growth and invasion in-vitro. Additionally, subcutaneous (SC) mouse model with these two inhibitors also showed effective inhibition of tumor growth in-vivo. Therefore, RalA is a key player in the biology and dispersal of ovarian cancer cells and is a promising target for the development of novel therapeutics. To the best of our knowledge, the role of RalA in ovarian cancer tumorigenesis has not been documented before, therefore our strategy in targeting RalA for treatment of ovarian cancer merits highly in terms of novelty. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-16. doi:10.1158/1538-7445.AM2011-LB-16

Pressure effects on the GTPase activity of brain membrane G proteins of deep-living marine fishes

In marine fishes, heterotrimeric guanyl nucleotide binding proteins (G proteins), which couple cell surface membrane receptors to their effector elements, are sensitive to hydrostatic pressure. The intrinsic high affinity GTPase activity of the α subunits of G proteins in three signaling systems coupled to adenylyl cyclase, the A 1 adenosine receptor, the muscarinic cholinergic receptor and the β-adrenergic receptor, was tested at pressures up to 340 atm. Brain membrane preparations from four members of the deep-sea teleost fish family Macrouridae were studied. Coryphaenoides armatus, C. filifer, C. rupestris and Macrourus berglax have depth distributions which together span 100–5810 m. Increased pressure inhibited basal GTPase activity only in M. berglax, which of the four species has the shallowest center of abundance. Increased hydrostatic pressure did not alter the response of GTPase activity to the β-adrenergic receptor agonist isoproterenol. Increased pressure decreased the stimulation of GTPase activity by the A 1 adenosine receptor agonist cyclopentyladenosine (CPA) in C. armatus and M. berglax, and by the muscarinic cholinergic receptor agonist carbamyl choline in C. armatus, C. filifer and M. berglax. Decreased agonist-stimulation of the GTPase activity at elevated pressure may result from pressure-induced changes in conformational states or inhibition of agonist binding. The binding of the non-hydrolyzable GTP analog guanosine 5′-[γ-thio]triphosphate (GTP[S]) in response to CPA was determined at 5 °C and atmospheric pressure. Six macrourid species and a morid were studied. The halftime ( t 1/2) values for GTP[S] binding, ranging from 20.8 to 40.9 min, are similar to values previously reported for two other cold-adapted fishes.

Anemia: A Cause of Intolerance to Thyroxine Sodium

Anemia: A Cause of Intolerance to Thyroxine Sodium

Recent bioorganic studies on rhodopsin and visual transduction.

Rhodopsin, the pigment responsible for vision in animals, insect and fish is a typical G protein (guanyl-nucleotide binding protein) consisting of seven transmembrane alpha helices and their interconnecting extramembrane loops. In the case of bovine rhodopsin, the best studied of the visual pigments, the chromophore is 11-cis retinal attached to the terminal amino group of Lys296 through a protonated Schiff base linkage. Photoaffinity labeling with a 3-diazo-4-oxo-retinoid shows that C-3 of the ionone ring moiety is close to Trp265 in helix F (VI) in dark inactivated rhodopsin. Irradiation causes a cis to trans isomerization of the 11-cis double bond giving rise to the highly strained intermediate bathorhodopsin. This undergoes a series of thermal relaxation through lumi-, meta-I and meta-II intermediates after which the retinal chromophore is expelled from the opsin binding pocket. Photoaffinity labeling performed with 3-diazo-4-oxoretinal at -196 degrees C for batho-, -80 degrees C for lumi-, -40 degrees C for meta-I, and 0 degrees C for meta-II rhodopsin showed that in bathorhodopsin the ring is still close to Trp265. However, in lumi-, meta-I and meta-II intermediates crosslinking occurs unexpectedly at A169 in helix D (IV). This shows that large movements in the helical arrangements and a flip over of the ring moiety accompanies the transduction (or bleaching) process. These changes in retinal/opsin interactions are necessarily accompanied by movements of the extramembrane loops, which in turn lead to activation of the G protein residing in the cytoplasmic side. Of the numerous G protein coupled receptors, this is the first time that the outline of transduction pathway has been clarified.

The effects of hydrostatic pressure on pertussis toxin-catalyzed ribosylation of G proteins from deep-living macrourid fishes

To test the effects of hydrostatic pressure on the coupling of receptors to guanyl nucleotide binding reglatory proteins (G proteins) in transmembrane signaling, pertussis toxin (PTX)-catalyzed [ 32P]ADP-ribosylation was used to probe the guanyl nucleotide-binding proteins G i and G o in brain membranes from four marine teleosts. These macrourids, Coryphaenoides pectoralis, Coryphaenoides cinereus, Coryphaenoides filifer and Coryphaenoides armatus, span depths from 200 to 5400 m. Pertussis toxin specifically labelled proteins of 39–41 kDa. The PTX-catalyzed [ 32P]ADP-ribosylation reaction was linear for 7 h. Added guanyl nucleotides (guanosine 5′-diphosphate (GDP) and guanosine 5′- O-(3-thiotriphosphate)(GTP[S])) at concentrations up to 1000 μM did not affect ribosylation at atmospheric pressure. Under basal conditions the G i/G o protein population appears to be uncoupled from receptors and bound with GDP. Pressures up to 476 atm were tested in the absence and presence of added guanyl nucleotides, 100 μM GDP and 100 μM GTP[S]. [ 32P]ADP-ribosylation in brain membranes from the deeper-occurring C. cinereus, C. filifer and C. armatus was not inhibited by increased pressure in the presence of 100 μM GDP. Increasing pressure decreased ribosylation in brain membranes of C. pectoralis. In the presence of 100 μM GTP[S], increased pressure inhibited ribosylation in all species. Pressure appears to enhance the efficacy of GTP[S] in dissociating the heterotrimeric holoprotein.

A role for guanyl nucleotide-binding regulatory protein beta- and gamma-subunits in the expression of the adrenocorticotropin receptor.

Mutant Y1 mouse adrenocortical tumor cells, isolated on the basis of their resistance to the growth-inhibitory effects of forskolin, arise from single mutational events. These mutants present complex phenotypes in which the activity of Gbeta/gamma is impaired, ACTH receptor gene expression is markedly diminished, and ACTH-responsive adenylyl cyclase activity is lost. In this study, we have tested the hypothesis that the impairment in Gbeta/gamma activity is responsible for the loss of ACTH receptor gene expression and ACTH-responsive adenylyl cyclase activity. Transfection of one of the mutant clones with expression vectors encoding either Gbeta1 or Gbeta2 together with Ggamma2 increased ACTH receptor expression and restored ACTH-responsive adenylyl cyclase activity. Interestingly, either Gbeta2 or Ggamma2 alone was effective. These results thus support the hypothesis that the impairment in Gbeta/gamma activity is responsible for the loss of ACTH receptor expression. A luciferase reporter plasmid driven by the proximal promoter region of the mouse ACTH receptor gene was expressed poorly in the mutants compared with parental Y1 cells, suggesting that the Gbeta/gamma defect compromised transcriptional activity at the proximal promoter region of the ACTH receptor gene.

G Protein-Mediated Inhibition of Neuronal Migration Requires Calcium Influx

Neuronal migration is an essential feature of the developing nervous system, but the intracellular signaling mechanisms that regulate this process are poorly understood. During the formation of the enteric nervous system (ENS) in the moth Manduca sexta, the migration of an identified set of neurons (the EP cells) is regulated in part by the heterotrimeric guanyl-nucleotide binding protein (G protein) Goalpha. Using an in vivo culture preparation for developing embryos that allows direct access to the ENS, we have shown that EP cell migration is similarly regulated by intracellular Ca2+; treatments that increased intracellular Ca2+ inhibited the migratory process, whereas buffering intracellular Ca2+ induced aberrant migration onto inappropriate pathways. Imaging the spontaneous changes in intracellular Ca2+ within individual EP cells showed that actively migrating neurons exhibited only small fluctuations in intracellular Ca2+. In contrast, neurons that had reached the end of migration displayed large, transient Ca2+ spikes. Similar Ca2+ spikes were induced in the EP cells by G protein stimulation, an effect that was reversed by removal of external Ca2+. Stimulation of Go in individual EP cells (by injection of either activated Goalpha subunits or mastoparan) also inhibited migration in a Ca2+-dependent manner. These results suggest that the regulation of neuronal migration by G proteins involves a Ca2+-dependent process requiring Ca2+ influx.

Redistribution of Gq/11α in the Pituitary Gonadotrope in Response to a Gonadotropin-Releasing Hormone Agonist*

In the present study, we took advantage of high-resolution multilaser confocal microscopy to examine the distribution of the alpha-subunit of the guanyl nucleotide binding protein subfamily G(q/11) (G(q/11)alpha). Dispersed cultures of pituitary cells were prepared from female weanling rats, fixed, permeabilized, and then stained with monoclonal antiserum (mouse) to the gonadotrope-specific form of secretogranin (SIIp), which was then tagged with Texas Red. Accordingly, the subpopulation of gonadotropes (approximately 15% of total cells) could be identified against a background of other pituitary cell types. G(q/11)alpha was localized with antiserum made in rabbit, then tagged with fluorescein. Hoechst 33258 nuclear stain was also used in some experiments for topological reference. The data indicate localization of the G(q/11)alpha in a cellular region near the plasma membrane and external to the border of the layer occupied by secretory granules. In the absence of activation, there were an average of six clusters of G(q/11)alpha in a section 1 microm thick and through the center of the cell. This corresponds to an average of 60 clusters per cell, assuming a mean gonadotrope diameter of 10 microm. Following continuous treatment with 0.1 microg/ml Buserelin, a metabolically stable GnRH agonist, the average number of clusters increased to 200/cell after 40 min and remained approximately constant for 120 min. This increase was blocked by the protein synthesis inhibitor, cycloheximide. In response to Buserelin, there was an additional increase in the number of clusters inside the cell in the area occupied by the secretory granules and in the perinuclear area. Prolonged (24 h) treatment with Buserelin, sufficient to provoke the onset of desensitization, did not significantly change total numbers of G(q/11)alpha clusters, although more were located in the peripheral compartment, an increase that occurred at the expense of the cytoplasmic compartment. Redistribution of the G(q/11)alpha family may be functionally significant, because this moiety may be rate limiting at the site of regulation of signal transduction.

Acute effects of progesterone on glucose metabolism in rat adipocytes: Are they modulated by endogenous adenosine?

Progesterone rapidly inhibits glucose oxidation of isolated rat adipocytes. Because this inhibition is triggered by endogenous adenosine, the present study was designed to examine the effect of the steroid on cyclic adenosine monophosphate (cAMP) accumulation, its relation to lipolysis, and the possible participation of adenosine. The results strongly indicate that physiological concentrations of progesterone increase the release of adenosine by isolated adipocytes, with maximal release at the end of a 20-minute incubation. Progesterone decreased both cAMP levels and lipolysis in quiescent adipocytes or in adipocytes stimulated by isoproterenol. The increase of endogenous adenosine may explain the decline of cAMP and glycerol levels observed with progesterone. The effects of the steroid on lipolysis disappeared when adenosine was hydrolyzed by adenosine deaminase (ADA). On the other hand, in the absence of endogenous adenosine, the effect of progesterone on the cAMP level was decreased only in isoproterenol-stimulated cells. The inhibitory effects of progesterone on cAMP and glycerol production seem not to be related directly to the adenosine A 1 receptor, for selective A 1 receptor antagonists (8-cyclopentyl-1,3-dipropylxanthine [DPCPX] and CP 68,247) did not counteract these effects. However, mechanisms mediated by guanyl nucleotide binding proteins cannot be excluded. The decrease of cAMP and of lipolysis may be related to a stimulation of phosphodiesterases (PDEs). When PDEs I (Ca 2+ — calmodulin-regulated PDE family) were blocked by a selective inhibitor (CP 41,757), the progesterone inhibitory effect persisted, suggesting that PDEs I are not regulated by the steroid. On the other hand, the progesterone effect on cAMP accumulation but not on lipolysis disappeared in the presence of a selective inhibitor of the PDE IV family (cAMP-dependent-specific family), Ro 20-1724. When the specific inhibitor of PDE I or PDE IV was combined with ADA, the progesterone effect on cAMP disappeared. Taken together, these results suggest that the progesterone inhibitory action on cAMP levels was not mediated through A 1 receptors or through activation of PDE I, but may be related to PDE IV activities. The progesterone effect on lipolysis seemed not to be directly related to changes in cAMP levels; an effect on PDE III activities in relation with the increase of adenosine release cannot be excluded.

Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

The neurohypophyseal nonapeptide oxytocin (OT) is the main hormone responsible for the initiation of labor; uterus contraction can be enhanced by application of oxytocin or suppressed by oxytocin antagonists. By transfer of domains from the G protein-coupled OT receptor into the related V2 vasopressin receptor, chimeric "gain in function" V2/OT receptors were produced that were able to bind either OT receptor agonists or a competitive peptide antagonist with high affinity. The binding site for the OT antagonist d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr9]vasotocin was found to be formed by transmembrane helices 1, 2, and 7 with a major contribution to binding affinity by the upper part of helix 7. These transmembrane receptor regions could be excluded from participating in OT binding. For agonist binding and selectivity the first three extracellular receptor domains were most important. The interaction of the N-terminal domain and of the first extracellular loop of the OT receptor with the linear C-terminal tripeptidic part of oxytocin was demonstrated. Furthermore, the second extracellular loop of the OT receptor could be identified to interact with the cyclic hormone part. These three domains contribute to OT binding by synergistic interaction with oxytocin but not with the competitive antagonist. Our results provide evidence for the existence of separate domains and different conformations of a peptide hormone receptor involved in binding and selectivity for agonists and peptide antagonists.

CDNA for the β1 subunit of guanyl nucleotide-binding regulatory proteins from mouse adrenal glands

cDNA encoding the β1 subunit of guanyl nucleotide binding regulatory proteins was isolated from a mouse adrenal cDNA library. The coding region was 90% identical to human and bovine β1 at the cDNA level and 100% identical at the protein level. In the 5′ untranslated region, two sequence variants were isolated that differed by the presence or absence of a 49 base pair insert presumed to arise from alternative splicing.

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: MOLECULAR MECHANISMS OF ANGIOTENSIN II (AT1a) RECEPTOR ENDOCYTOSIS

1. Angiotensin II (AngII) initiates a variety of cellular responses through activation of type 1 (AT(1); with subtypes AT(1a) and AT(1b) ) and type 2 (AT(2) ) cell surface angiotensin receptors. Both AT(1) and AT(2) receptors couple to heterotrimeric guanyl nucleotide binding proteins (G-proteins) and generate intracellular signals following recognition of extracellular AngII, but only AT(1) is targeted for the rapid ligand-stimulated endocytosis (internalization) typical of many plasma membrane receptors. 2. AT(1) endocytosis proceeds through clathrin-coated pits and is independent of G-protein coupling which predicts that the AngII-AT(1) receptor complex attains a conformation necessary for interaction with the endocytotic machinery, but separate from receptor signalling activation. 3. The function of AT(1) endocytosis and the reason for the disparity between AT(1) and AT(2) endocytosis is not fully appreciated, but the latter probably reflects differences in the primary amino acid sequence of these two receptor types. 4. For many receptors that undergo internalization, it has been established that internalization motifs (2-6 amino acids, often incorporating crucial tyrosine and hydrophobic amino acids) within the cytoplasmic regions of the receptor mediate the selective recruitment of activated receptors into clathrin-coated pits and vesicles. 5. Mutagenesis studies on the AT(1a) receptor, aimed at identifying such motifs, reveal that sites within the third cytoplasmic loop and the cytoplasmic carboxyl terminal region are important for AngII-stimulated AT(1a) receptor endocytosis.

Palmitoylation of Endothelin Receptor A: DIFFERENTIAL MODULATION OF SIGNAL TRANSDUCTION ACTIVITY BY POST-TRANSLATIONAL MODIFICATION

Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins. Here we demonstrate that the human endothelin receptor A (ETA) incorporates [3H]palmitate. Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ETA into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ETA mutants was essentially unchanged as compared to the palmitoylated wild type ETA suggesting that the replacement of the cysteine residues did not alter the overall structure of the receptor. Furthermore, the ligand-induced stimulation of adenylyl cyclase by the mutant ETA was unaffected by the mutation. In contrast, the mutated non-palmitoylated receptors but not the wild type receptor failed to stimulate phosphatidylinositol hydrolysis by phospholipase C activation upon challenge by endothelin-1. Furthermore, the mutant receptors failed to stimulate the ligand-induced transient increase in the cytoplasmic calcium seen with the wild type ETA. Endothelin-1 induced mitogenic stimuli via the wild type receptors but not through the mutated receptors suggesting an important role for phospholipase C in this signal transduction pathway. The differential regulation of distinct signal transduction pathways by post-translational modification suggests that palmitoylation of the ETA provides a novel mechanism of modulating ETA receptor activity.

REVIEW ■ : ATP-sensitive Potassium Channels: Diverse Functions in the Central Nervous System

ATP-sensitive potassium channels open when cytoplasmic levels of ATP drop, thus linking membrane potential to the metabolic state of the cell. Cloning studies have suggested that these channels are related structurally to the inward rectifier family of potassium channels, with two putative membrane-spanning regions. Sulfonylurea drugs, which are used in the treatment of diabetes, inhibit these channels by binding to an associated membrane protein. Other drugs, including some vasodilators, activate ATP-sensitive potassium channels. Diverse neurotransmitter and hormone receptors can modulate these channels, in some cases through interactions with guanyl nucleotide binding proteins. There appear to be multiple subtypes of these channels, differing in electrical properties as well as in drug sensitivities. In the brain, these channels appear to play a role in mediating satiety after feeding. They also function in neurons to protect against excitotoxicity, by counteracting the membrane depolarization associated with metabolic stress. Brain dopamine receptors appear to modulate a novel subtype of ATP-sensitive potassium channel. The association of dopamine receptors with a mechanism involved in protection against neurodegeneration may have implications for the causes of diseases in which dopaminergic regions of brain undergo structural changes, possibly including schizophrenia. NEUROSCIENTIST 2:145-152, 1996

A Developmental Role for the Heterotrimeric G Protein Goαin a Migratory Population of Embryonic Neurons

The heterotrimeric G proteins are an extended family of guanyl nucleotide-binding proteins that serve essential functions in the mature nervous system but whose contributions to neuronal development remain poorly understood. We have investigated the potential role of one specific G protein, Goα, in the control of neuronal migration. During embryogenesis of the moth,Manduca sexta,an identified population of undifferentiated neurons (the EP cells) migrate along sets of visceral muscle bands to form part of the enteric nervous system. Previously, immunohistochemical studies indicated the presence of Goα-related proteins in the EP cells during migration. We have now verified this result, using probes derived from the Goαgene inManduca.A clone containing the full-length coding domain for Goαwas sequenced from aManducacDNA library; digoxigenin-labeled probes were then made from this clone and used to examine the developmental expression of the Goαgene during embryogenesis. Goα-specific transcripts could first be detected in the EP cells several hours before the onset of their migration. The level of Goαexpression in all of the EP cells continued to increase during migration, but subsequently was down-regulated in a subset of the postmigratory neurons at the time of their terminal differentiation. This pattern of regulated expression is consistent with the distribution of Goα-related protein in the EP cells. We also used a semi-intact culture preparation of staged embryos to investigate the effects of G protein-specific toxins on the migratory process. Intracellular injections of the wasp toxin mastoparan, a specific activator of Goα- and Giα-related proteins, inhibited the migration of individual EP cells. Injections of pertussis toxin (an inhibitor of Goαand Giα) or cholera toxin (a selective activator of Gsα) had no effect on migration, although pertussis toxin treatments did cause a measurable increase in the subsequent outgrowth of axonal processes. However, co-injection of mastoparan with pertussis toxin blocked the inhibitory effects of mastoparan alone. These results suggest that Goα-coupled signaling events within the EP cells may down-regulate their migratory behavior, possibly in response to inhibitory cues that normally guide migration in the developing embryo.

Cooperativity Manifest in the Binding Properties of Purified Cardiac Muscarinic Receptors

Muscarinic receptors were solubilized from porcine atria in digitonin-cholate and were purified by chromatography on DEAE-Sepharose and 3-(2'-aminobenzhydryloxy)tropane-Sepharose. The product identified on Western blots migrated with an apparent molecular mass of 60-75 kDa, with additional bands indicative of homotrimers (190 kDa) and homotetramers (240 kDa). Receptor eluted from the affinity column was accompanied by a mixture of guanyl nucleotide-binding proteins (G-proteins) identified on Western blots as Gi1/2, G(o), Gq/11, and Gs (preparation M2G); the G-proteins were largely removed by further processing on hydroxyapatite (preparation M2). Solubilized purified receptors bound muscarinic ligands in an apparently cooperative manner. In studies at equilibrium, the antagonists [3H]AF-DX 384, N-[3H]methylscopolamine (NMS), and [3H]quinuclidinylbenzilate (QNB) revealed Hill coefficients between about 0.8 and 1.2. Also, the apparent capacity for [3H]QNB exceeded that for [3H]AF-DX 384 and [3H]NMS by about 1.5-fold in M2 and by 2-fold in M2G. Binding to M2G at high concentrations of [3H]QNB was fully inhibited by unlabeled NMS, which therefore affected sites not labeled at similar concentrations of [3H]NMS. Oxotremorine-M displayed a biphasic inhibitory effect on the binding of [3H]AF-DX 384 in M2 and M2G, suggesting that multiple states of affinity are intrinsic to the receptor; 5'-guanylylimidodiphosphate was without appreciable effect in M2 but resulted in a bell-shaped binding profile for the agonist in M2G. All of the data can be described in terms of cooperative interactions within a receptor that is at least tetravalent and presumably an oligomer. In the context of the model, copurifying G-proteins and guanyl nucleotides serve to regulate the degree of cooperativity between successive equivalents of muscarinic ligands.

Mastoparan activation of dopamine-modulated K+ channels on rat striatal neurons.

Coupling mechanisms between a D2-like dopamine receptor and an 85 pS K+ channel on freshly dissociated rat caudate-putamen neurons were studied using cell-attached patch-clamp electrophysiology. Channel currents were observed in the absence of dopamine receptor agonists when mastoparan, an activator of certain guanyl nucleotide binding proteins (G-proteins), was applied via the patch pipette. Channel current-voltage relationships and open probabilities observed with mastoparan were indistinguishable from those obtained with dopaminergic agonists. Alamethicin, which mimics the membrane-perturbing but not the G-protein activating properties of mastoparan, did not activate the channel, suggesting that non-specific effects of mastoparan were unlikely to account for this effect. Our results indicate that coupling between the D2 dopamine receptor and this K+ channel may involve a mastoparan-sensitive G-protein.

Developmental expression of heterotrimeric G proteins in the nervous system of Manduca sexta

The heterotrimeric G proteins are a conserved family of guanyl nucleotide-binding proteins that appear in all eukaryotic cells but whose developmental functions are largely unknown. We have examined the developmental expression of representative G proteins in the developing nervous system of the moth Manduca sexta. Using affinity-purified antisera against different G alpha subunits, we found that each of the G proteins exhibited distinctive patterns of expression within the developing central nervous system (CNS), and that these patterns underwent progressive phases of spatial and temporal regulation that corresponded to specific aspects of neuronal differentiation. Several of the G proteins examined (including Gs alpha and G(o) alpha) were expressed in an apparently ubiquitous manner in all neurons, but other proteins (including Gi alpha) were ultimately confined to a more restricted subset of cells in the mature CNS. Although most of the G proteins examined could be detected within the central ganglia, only G(o) alpha-related proteins were seen in the developing peripheral nerves; manipulations of G protein activity in cultured embryos suggested that this class of G protein may contribute to the regulation of neuronal motility during axonal outgrowth. G(o) alpha-related proteins were also localized to the developing axons and terminals of the developing adult limb during metamorphosis. These intracellular signaling molecules may, therefore, play similar developmental roles in both the embryonic and postembryonic nervous system.

Absence of reduced platelet adenylate cyclase activity in Vietnam veterans with PTSD

Many studies have been devoted to an examination of radioligand receptor binding and postreceptor signal transduction in peripheral cells obtained from patients suffering from mood or anxiety disorders. Postreceptor signal transduction has been investigated by the use of agents that interact with the adenylate cyclase system at points distal to the receptor, such as aluminum chloride (AIC13) and sodium fluoride (NaF), which affect the guanyl nucleotidebinding (G) protein, and forskolin, which affects the catalytic (C) unit of the enzyme. Post-traumatic stress disorder (PTSD) is a member of the group of anxiety disorders which also includes generalized anxiety disorder, panic disorder, and obsessive-compulsive disorder. PTSD also shares phenomenological features with depression (Sierles et al 1983). Abnormalities in adenylate cyclase activity have been demonstrated in peripheral cells derived both from panic disorder patients (Charney et al 1989) and depressed patients (Ebstein et a11988; Kanof et al 1988). Recent evidence has indicated possible abnormalities at both the receptor and postreeeptor levels in platelets from PTSD patients. Perry et al (1987) found a reduction in alpha-2-adrenergic receptors in 11 Vietnam veterans with PTSD relative to agematched normal controls. This was due to a relative loss of sites labeled with low affinity by the antagonist 3H-rauwolscine, representing those sites coupled via the inhibitory G protein Gi to adenylate cyclase. The involvement of adrenergic receptors in PTSD has been recently reviewed (Lerer et al 1994). In a series of studies, our laboratory has investigated platelet adenylate cyclase in PTSD with mixed results. A first study (Lerer