High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma.

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

Gqα/G11α deficiency in dorsomedial hypothalamus leads to obesity resulting from decreased energy expenditure and impaired sympathetic nerve activity.

The G-protein subunits Gqα and G11α (Gq/11α) couple receptors to phospholipase C, leading to increased intracellular calcium. In this study we investigated the consequences of Gq/11α deficiency in the dorsomedial hypothalamus (DMH), a critical site for the control of energy homeostasis. Mice with DMH-specific deletion of Gq/11α (DMHGq/11KO) were generated by stereotaxic injection of adeno-associated virus (AAV)-Cre-green fluorescent protein (GFP) into the DMH of Gqαflox/flox:G11α-/- mice. Compared with control mice that received DMH injection of AAV-GFP, DMHGq/11KO mice developed obesity associated with reduced energy expenditure without significant changes in food intake or physical activity. DMHGq/11KO mice showed no defects in the ability of the melanocortin agonist melanotan II to acutely stimulate energy expenditure or to inhibit food intake. At room temperature (22°C), DMHGq/11KO mice showed reduced sympathetic nervous system activity in brown adipose tissue (BAT) and heart, accompanied with decreased basal BAT uncoupling protein 1 (Ucp1) gene expression and lower heart rates. These mice were cold intolerant when acutely exposed to cold (6°C for 5 h) and had decreased cold-stimulated BAT Ucp1 gene expression. DMHGq/11KO mice also failed to adapt to gradually declining ambient temperatures and to develop adipocyte browning in inguinal white adipose tissue although their BAT Ucp1 was proportionally stimulated. Consistent with impaired cold-induced thermogenesis, the onset of obesity in DMHGq/11KO mice was significantly delayed when housed under thermoneutral conditions (30°C). Thus our results show that Gqα and G11α in the DMH are required for the control of energy homeostasis by stimulating energy expenditure and thermoregulation.NEW & NOTEWORTHY This paper demonstrates that signaling within the dorsomedial hypothalamus via the G proteins Gqα and G11α, which couple cell surface receptors to the stimulation of phospholipase C, is critical for regulation of energy expenditure, thermoregulation by brown adipose tissue and the induction of white adipose tissue browning.

Uveal melanoma: physiopathology and new in situ-specific therapies.

Uveal melanoma is the most common primary intraocular tumor in adults. It can arise from melanocytes in the anterior (iris) or posterior uveal tract (choroid and ciliary body). Uveal melanoma has a particular molecular pathogenesis, being characterized by specific chromosome alterations and gene mutations (e.g., GNAQ/GNA11; BAP1), which are considered promising targets for molecular therapy. Primary treatment of uveal melanoma includes radiotherapy (brachytherapy and charged-particle therapy), phototherapy (photocoagulation, transpupillary thermal therapy, and photodynamic therapy) and surgery (local resection, enucleation and exenteration). Approximately half of patients with uveal melanoma will, however, develop metastasis, especially in the liver. The treatment of metastatic uveal melanoma includes systemic chemotherapy, immunotherapy and molecular targeted therapy. Liver-directed therapies, such as resection, chemoembolization, immunoembolization, radioembolization, isolated hepatic perfusion and percutaneous hepatic perfusion, are also available to treat metastatic uveal melanoma. Several clinical trials are being developed to study new therapeutic options to treat uveal melanoma, mainly for those with identified liver metastases. The present work discusses the physiopathology and new in situ-specific therapies for the treatment of uveal melanoma.

Tanycytes control the hormonal output of the hypothalamic-pituitary-thyroid axis.

The hypothalamic-pituitary-thyroid (HPT) axis maintains circulating thyroid hormone levels in a narrow physiological range. As axons containing thyrotropin-releasing hormone (TRH) terminate on hypothalamic tanycytes, these specialized glial cells have been suggested to influence the activity of the HPT axis, but their exact role remained enigmatic. Here, we demonstrate that stimulation of the TRH receptor 1 increases intracellular calcium in tanycytes of the median eminence via Gαq/11 proteins. Activation of Gαq/11 pathways increases the size of tanycyte endfeet that shield pituitary vessels and induces the activity of the TRH-degrading ectoenzyme. Both mechanisms may limit the TRH release to the pituitary. Indeed, blocking TRH signaling in tanycytes by deleting Gαq/11 proteins in vivo enhances the response of the HPT axis to the chemogenetic activation of TRH neurons. In conclusion, we identify new TRH- and Gαq/11-dependent mechanisms in the median eminence by which tanycytes control the activity of the HPT axis.The hypothalamic-pituitary-thyroid (HPT) axis regulates a wide range of physiological processes. Here the authors show that hypothalamic tanycytes play a role in the homeostatic regulation of the HPT axis; activation of TRH signaling in tanycytes elevates their intracellular Ca2+ via Gαq/11 pathway, ultimately resulting in reduced TRH release into the pituitary vessels.

Impact of obesity on taste receptor expression in extra-oral tissues: emphasis on hypothalamus and brainstem.

Sweet perception promotes food intake, whereas that of bitterness is inhibitory. Surprisingly, the expression of sweet G protein-coupled taste receptor (GPCTR) subunits (T1R2 and T1R3) and bitter GPCTRs (T2R116, T2R118, T2R138 and T2R104), as well as the α-subunits of the associated signalling complex (αGustducin, Gα14 and αTransducin), in oral and extra-oral tissues from lean and obese mice, remains poorly characterized. We focused on the impact of obesity on taste receptor expression in brain areas involved in energy homeostasis, namely the hypothalamus and brainstem. We demonstrate that many of the GPCTRs and α-subunits are co-expressed in these tissues and that obesity decreases expression of T1R3, T2R116, Gα14, αTrans and TRPM5. In vitro high levels of glucose caused a prominent down-regulation of T1R2 and Gα14 expression in cultured hypothalamic neuronal cells, leptin caused a transient down-regulation of T1R2 and T1R3 expression. Intriguingly, expression differences were also observed in other extra-oral tissues of lean and obese mice, most strikingly in the duodenum where obesity reduced the expression of most bitter and sweet receptors. In conclusion, obesity influences components of sweet and bitter taste sensing in the duodenum as well as regions of the mouse brain involved in energy homeostasis, including hypothalamus and brainstem.

Phasic and Tonic mGlu7 Receptor Activity Modulates the Thalamocortical Network.

Mutation of the metabotropic glutamate receptor type 7 (mGlu7) induces absence-like epileptic seizures, but its precise role in the somatosensory thalamocortical network remains unknown. By combining electrophysiological recordings, optogenetics, and pharmacology, we dissected the contribution of the mGlu7 receptor at mouse thalamic synapses. We found that mGlu7 is functionally expressed at both glutamatergic and GABAergic synapses, where it can inhibit neurotransmission and regulate short-term plasticity. These effects depend on the PDZ-ligand of the receptor, as they are lost in mutant mice. Interestingly, the very low affinity of mGlu7 receptors for glutamate raises the question of how it can be activated, namely at GABAergic synapses and in basal conditions. Inactivation of the receptor activity with the mGlu7 negative allosteric modulator (NAM), ADX71743, enhances thalamic synaptic transmission. In vivo administration of the NAM induces a lethargic state with spindle and/or spike-and-wave discharges accompanied by a behavioral arrest typical of absence epileptic seizures. This provides evidence for mGlu7 receptor-mediated tonic modulation of a physiological function in vivo preventing synchronous and potentially pathological oscillations.

Effect of yizhitongxuan decoction on learning and memory ability, Gaq/11 expression and Na(+)-K(+)-ATP enzyme activity in rat models of Alzheimer's disease.

OBJECTIVE: To study the effects of Yizhitongxuan decoction on learning and memory abilities, Gaq/11 expression and Na(+)-K(+)-ATP enzyme activity in rat models of Alzheimer's disease (AD) caused by injecting Abeta25-35 into the hippocampus. METHODS: Ninety male Wistar rats (age > or = 10 months) were selected and injected with Abeta25-35 into their hippocampi to establish model animals, which were randomly divided into six groups including a sham-operated group (blank group), a model group, a donepezil HCL group (Western Medicine group), and a high/general/dilute concentrations of Yizhitongxuan decoction groups (TCM I II III group). The Morris water maze was used to examine the learning and memory abilities of rats in each group by place navigation and spatial probe tests. Then, the rats were sacrificed to collect the hippocampi for biochemical tests, using western blotting to detect the expression of Gaq/11 and an ultramicro Na(+)-K(+)-ATP enzyme kit to measure Na(+)-K(+)-ATP enzyme activity. RESULTS: Yizhitongxuan decoction improved model rats' learning and memory abilities, and increased the expression of Gaq/11 in the hippocampus and the level of Na(+)-K(+)-ATP enzyme activity in brain tissue. CONCLUSION: Yizhitongxuan decoction could improve model rats' learning and memory abilities, and had a regulating effect on the expression of Gaq/11 and Na(+)-K(+)-ATP enzyme activity.

GNAQ/11 mutations in uveal melanoma: is YAP the key to targeted therapy?

GNAQ and GNA11 are frequently mutated in uveal melanoma, but they remain difficult therapeutic targets. In this issue of Cancer Cell, Feng and colleagues and Yu and colleagues demonstrate that the oncogenic activity of mutant GNAQ/11 is mediated at least in part through YAP, potentially uncovering a new therapeutic strategy.

Chronic activation of a designer G(q)-coupled receptor improves ß cell function.

Type 2 diabetes (T2D) has emerged as a major threat to human health in most parts of the world. Therapeutic strategies aimed at improving pancreatic ß cell function are predicted to prove beneficial for the treatment of T2D. In the present study, we demonstrate that drug-mediated, chronic, and selective activation of ß cell G(q) signaling greatly improve ß cell function and glucose homeostasis in mice. These beneficial metabolic effects were accompanied by the enhanced expression of many genes critical for ß cell function, maintenance, and differentiation. By employing a combination of in vivo and in vitro approaches, we identified a novel ß cell pathway through which receptor-activated G(q) leads to the sequential activation of ERK1/2 and IRS2 signaling, thus triggering a series of events that greatly improve ß cell function. Importantly, we found that chronic stimulation of a designer G(q)-coupled receptor selectively expressed in ß cells prevented both streptozotocin-induced diabetes and the metabolic deficits associated with the consumption of a high-fat diet in mice. Since ß cells are endowed with numerous receptors that mediate their cellular effects via activation of G(q)-type G proteins, our findings provide a rational basis for the development of novel antidiabetic drugs targeting this class of receptors.

Substrate specificity of Pasteurella multocida toxin for α subunits of heterotrimeric G proteins.

Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gα(i2), it was demonstrated that the toxin deamidates an essential glutamine residue of the Gα(i2) subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G-protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gα(q/11), Gα(i1,2,3), and Gα(12/13) of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G-protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination.

Identification of the muscarinic pathway underlying cessation of sleep-related burst activity in rat thalamocortical relay neurons.

Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCß (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.

GPR54 regulates ERK1/2 activity and hypothalamic gene expression in a Gα(q/11) and ß-arrestin-dependent manner.

G protein-coupled receptor 54 (GPR54) is a G(q/11)-coupled 7 transmembrane-spanning receptor (7TMR). Activation of GPR54 by kisspeptin (Kp) stimulates PIP(2) hydrolysis, Ca(2+) mobilization and ERK1/2 MAPK phosphorylation. Kp and GPR54 are established regulators of the hypothalamic-pituitary-gonadal (HPG) axis and loss-of-function mutations in GPR54 are associated with an absence of puberty and hypogonadotropic hypogonadism, thus defining an important role of the Kp/GPR54 signaling system in reproductive function. Given the tremendous physiological and clinical importance of the Kp/GPR54 signaling system, we explored the contributions of the GPR54-coupled G(q/11) and ß-arrestin pathways on the activation of a major downstream signaling molecule, ERK, using G(q/11) and ß-arrestin knockout mouse embryonic fibroblasts. Our study revealed that GPR54 employs the G(q/11) and ß-arrestin-2 pathways in a co-dependent and temporally overlapping manner to positively regulate ERK activity and pERK nuclear localization. We also show that while ß-arrestin-2 potentiates GPR54 signaling to ERK, ß-arrestin-1 inhibits it. Our data also revealed that diminished ß-arrestin-1 and -2 expression in the GT1-7 GnRH hypothalamic neuronal cell line triggered distinct patterns of gene expression following Kp-10 treatment. Thus, ß-arrestin-1 and -2 also regulate distinct downstream responses in gene expression. Finally, we showed that GPR54, when uncoupled from the G(q/11) pathway, as is the case for several naturally occurring GPR54 mutants associated with hypogonadotropic hypogonadism, continues to regulate gene expression in a G protein-independent manner. These new and exciting findings add significantly to our mechanistic understanding of how this important receptor signals intracellularly in response to kisspeptin stimulation.

Disruption of adenylyl cyclase type V does not rescue the phenotype of cardiac-specific overexpression of Galphaq protein-induced cardiomyopathy.

Adenylyl cyclase (AC) is the principal effector molecule in the ß-adrenergic receptor pathway. AC(V) and AC(VI) are the two predominant isoforms in mammalian cardiac myocytes. The disparate roles among AC isoforms in cardiac hypertrophy and progression to heart failure have been under intense investigation. Specifically, the salutary effects resulting from the disruption of AC(V) have been established in multiple models of cardiomyopathy. It has been proposed that a continual activation of AC(V) through elevated levels of protein kinase C could play an integral role in mediating a hypertrophic response leading to progressive heart failure. Elevated protein kinase C is a common finding in heart failure and was demonstrated in murine cardiomyopathy from cardiac-specific overexpression of G(αq) protein. Here we assessed whether the disruption of AC(V) expression can improve cardiac function, limit electrophysiological remodeling, or improve survival in the G(αq) mouse model of heart failure. We directly tested the effects of gene-targeted disruption of AC(V) in transgenic mice with cardiac-specific overexpression of G(αq) protein using multiple techniques to assess the survival, cardiac function, as well as structural and electrical remodeling. Surprisingly, in contrast to other models of cardiomyopathy, AC(V) disruption did not improve survival or cardiac function, limit cardiac chamber dilation, halt hypertrophy, or prevent electrical remodeling in G(αq) transgenic mice. In conclusion, unlike other established models of cardiomyopathy, disrupting AC(V) expression in the G(αq) mouse model is insufficient to overcome several parallel pathophysiological processes leading to progressive heart failure.

Dehydroepiandrosterone administration or G{alpha}q overexpression induces {beta}-catenin/T-Cell factor signaling and growth via increasing association of estrogen receptor-{beta}/Dishevelled2 in androgen-independent prostate cancer cells.

beta-Catenin/T-cell factor signaling (beta-CTS) plays multiple critical roles in carcinogenesis and is blocked by androgens in androgen receptor (AR)-responsive prostate cancer (PrCa) cells, primarily via AR sequestration of beta-catenin from T-cell factor. Dehydroepiandrosterone (DHEA), often used as an over-the-counter nutritional supplement, is metabolized to androgens and estrogens in humans. The efficacy and safety of unregulated use of DHEA are unclear. We now report that DHEA induces beta-CTS via increasing association of estrogen receptor (ER)-beta with Dishevelled2 (Dvl2) in AR nonresponsive human PrCa DU145 cells, a line of androgen-independent PrCa (AiPC) cells. The induction is temporal, as assessed by measuring kinetics of the association of ERbeta/Dvl2, protein expression of the beta-CTS targeted genes, c-Myc and cyclin D1, and cell growth. However, in PC-3 cells, another human AiPC cell line, DHEA exerts no detectible effects, partly due to their lower expression of Galpha-subunits and DHEA down-regulation of ERbeta/Dvl2 association. When Galphaq is overexpressed in PC-3 cells, beta-CTS is constitutively induced, including increasing c-Myc and cyclin D1 protein expression. This effect involved increasing associations of Galphaq/Dvl2 and ERbeta/Dvl2 and promoted cell growth. These activities require ERbeta in DU-145 and PC-3 cells because they are blocked by ICI 182-780 treatment inactivating ERbeta, small interfering RNA administration depleting ERbeta, or AR overexpression arresting ERbeta. These data suggest that novel pathways activating beta-CTS play roles in the progression of AiPC. Although DHEA may enhance PrCa cell growth via androgenic or estrogenic pathways, the effects of DHEA administration on clinical prostate function remain to be determined.

Gq-dependent signaling upregulates COX2 in glomerular podocytes.

Accumulating evidence suggests that upregulation of cyclooxygenase 2 (COX2) in glomerular podocytes promotes podocyte injury. Because Gq signaling activates calcineurin and calcineurin-dependent mechanisms are known to mediate COX2 expression, this study investigated the role of Gqalpha in promoting COX2 expression in podocytes. A constitutively active Gq alpha subunit tagged with the TAT HIV protein sequence was introduced into an immortalized podocyte cell line by protein transduction. This stimulated inositol trisphosphate production, activated an nuclear factor of activated T cells-responsive reporter construct, and enhanced levels of both COX2 mRNA and protein compared with cells treated with a Gq protein lacking the TAT sequence. Induction of COX2 was associated with increased prostaglandin E(2) production and podocyte death, both of which were attenuated by selective COX2 inhibition. In vivo, levels of COX2 mRNA and protein were significantly enhanced in podocytes from transgenic mice that expressed podocyte-targeted constitutively active Gqalpha compared with nontransgenic littermates. These data suggest that Gq-dependent signaling cascades stimulate calcineurin and, in turn, upregulate COX2 mRNA and protein, increase eicosanoid production, and cause podocyte injury.

Functional human 5-HT6 receptor assay for high throughput screening of chemical ligands and binding proteins.

Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT(6) receptor (5-HT(6)R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT(6)R are poorly understood due to the lack of selective 5-HT(6)R ligands. In the present study, we examined functional coupling of the human 5-HT(6)R, 5-HT(7A)R, or 5-HT(7B)R with various Galpha-proteins (Galpha(15), Galpha(qs5), or Galpha(qG66Ds5)) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT(6)R with Galpha(qG66Ds5) produced the highest levels of Ca(2+) signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT(6)R HTS system (Z'-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT(6)R, we screened approximately 500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC(50) value of 6.3 microM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT(6)R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT(6)R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT(6)R interacting protein. Taken together, these results suggest that the 5-HT(6)R/Galpha(qG66Ds5) FDSS6000 system can be utilized to screen for selective 5-HT(6)R ligands and to examine any functional relationships between 5-HT(6)R and its binding proteins.

Muscarinic ACh receptor-mediated control of thalamic activity via G(q)/G (11)-family G-proteins.

A genetic knock out was used to determine the specific contribution of G(q)/G(11)-family G-proteins to the function of thalamocortical relay (TC) neurons. Disruption of Galpha(q) function in a conditional forebrain-specific Galpha(q)/Galpha(11)-double-deficient mouse line (Galpha(q)/Galpha(11)(-/-) had no effects on the resting membrane potential (V (rest)) and the amplitude of the standing outward current (I (SO)). Stimulation of muscarinic acetylcholine (ACh) receptors (mAChR; muscarine, 50 microM) induced a decrease in I (SO) amplitude in wild-type mice (36 +/- 4%, n = 5), a constitutive Galpha(11)-deficient mouse line (Galpha(11)(-/-; 36 +/- 3%, n = 8), and Galpha(q)/Galpha(11)(-/-) (11 +/- 2%, n = 16). Current-clamp recordings revealed a muscarine-induced positive shift in V (rest) of 23 +/- 2 mV (n = 6), 18 +/- 5 mV (n = 5), and 2 +/- 1 mV (n = 9) in wild type, Galpha(11)(-/-), and Galpha(q)/Galpha(11)(-/-), respectively. This depolarization was associated with a change in TC neuron activity from burst to tonic firing in wild type and Galpha(11)(-/-), but not in Galpha(q)/Galpha(11)(-/-). The use of specific antibodies and of pharmacological agents with preferred affinity points to the contribution of m(1)AChR and m(3)AChR. In conclusion, we present two novel aspects of the physiology of the thalamocortical system by demonstrating that the depolarization of TC neurons, which is induced by the action of transmitters of ascending brainstem fibers, is governed roughly equally by both m(1)AChR and m(3)AChR and is transduced by Galpha(q) but not by Galpha(11).

The pheromone biosynthesis activating neuropeptide (PBAN) receptor of Heliothis virescens: identification, functional expression, and structure-activity relationships of ligand analogs.

Pheromone biosynthesis activating neuropeptide (PBAN) promotes synthesis and release of sex pheromones in moths. We have identified and functionally expressed a PBAN receptor from Heliothis virescens (HevPBANR) and elucidated structure-activity relationships of PBAN analogs. Screening of a larval CNS cDNA library revealed three putative receptor subtypes and nucleotide sequence comparisons suggest that they are produced through alternative splicing at the 3'-end. RT-PCR amplified preferentially HevPBANR-C from female pheromone glands. CHO cells expressing HevPBANR-C are highly sensitive to PBAN and related analogs, especially those sharing the C-terminal pentapeptide core, FXPRLamide (X=T, S or V). Alanine replacements in the C-terminal hexapeptide (YFTPRLamide) revealed the relative importance of each residue in the active core as follows: R5>L6>F2>>P4>T3>>Y1. This study provides a framework for the rational design of PBANR-specific agonists and/or antagonists that could be exploited for disruption of reproductive function in agriculturally important insect pests.

A G(q/11)-coupled mutant histamine H(1) receptor F435A activated solely by synthetic ligands (RASSL).

Recently, G protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been introduced as new tools to study Galpha(i) signaling in vivo (1, 2). Also, Galpha(s)-coupled G protein-coupled receptors have been engineered to generate Galpha(s)-coupled RASSLs (3, 4). In this study, we exploited the differences in binding pockets between different classes of H(1) receptor agonists and identified the first Galpha(q/11)-coupled RASSL. The mutant human H(1) receptor F435A (6.55) combines a strongly decreased affinity (25-fold) and potency for the endogenous ligand histamine (200-fold) with improved affinities (54-fold) and potencies (2600-fold) for 2-phenylhistamines, a synthetic class of H(1) receptor agonists. Molecular dynamics simulations provided a mechanism for distinct agonist binding to both wild-type and F435A mutant H(1) receptors.

Loss of Gq/11 family G proteins in the nervous system causes pituitary somatotroph hypoplasia and dwarfism in mice.

Heterotrimeric G proteins of the Gq/11 family transduce signals from a variety of neurotransmitter and hormone receptors and have therefore been implicated in various functions of the nervous system. Using the Cre/loxP system, we generated mice which lack the genes coding for the alpha subunits of the two main members of the Gq/11 family, gnaq and gna11, selectively in neuronal and glial precursor cells. Mice with defective gnaq and gna11 genes were morphologically normal, but they died shortly after birth. Mice carrying a single gna11 allele survived the early postnatal period but died within 3 to 6 weeks as anorectic dwarfs. In these mice, postnatal proliferation of pituitary somatotroph cells was strongly impaired, and plasma growth hormone (GH) levels were reduced to 15%. Hypothalamic levels of GH-releasing hormone (GHRH), an important stimulator of somatotroph proliferation, were strongly decreased, and exogenous administration of GHRH restored normal proliferation. The hypothalamic effects of ghrelin, a regulator of GHRH production and food intake, were reduced in these mice, suggesting that an impairment of ghrelin receptor signaling might contribute to GHRH deficiency and abnormal eating behavior. Taken together, our findings show that Gq/11 signaling is required for normal hypothalamic function and that impairment of this signaling pathway causes somatotroph hypoplasia, dwarfism, and anorexia.