Protein Gamma Subunits Research Articles

Overcoming adaptive resistance in AML by synergistically targeting FOXO3A-GNG7-mTOR axis with FOXO3A inhibitor Gardenoside and rapamycin

Therapeutic targeting FOXO3A (a forkhead transcription factor) represents a promising strategy to suppress acute myeloid leukemia (AML). However, the effective inhibitors that target FOXO3A are lacking and the adaptive response signaling weakens the cytotoxic effect of FOXO3A depletion on AML cells. Here, we show that FOXO3A deficiency induces a compensatory response involved in the reactive activation of mTOR that leads to signaling rebound and adaptive resistance. Mitochondrial metabolism acts downstream of mTOR to provoke activation of JNK/c-JUN via reactive oxygen species (ROS). At the molecular level, FOXO3A directly binds to the promoter of G protein gamma subunit 7 (GNG7) and preserves its expression, while GNG7 interacts with mTOR and restricts phosphorylated activation of mTOR. Consequently, combinatorial inhibition of FOXO3A and mTOR show a synergistic cytotoxic effect on AML cells and prolongs survival in a mouse model of AML. Through a structure-based virtual screening, we report one potent small-molecule FOXO3A inhibitor (Gardenoside) that exhibits a strong effect of anti-FOXO3A DNA binding. Gardenoside synergizes with rapamycin to substantially reduce tumor burden and extend survival in AML patient-derived xenograft model. These results demonstrate that mTOR can mediate adaptive resistance to FOXO3A inhibition and validate a combinatorial approach for treating AML.

N‐terminal Intrinsic Disorder of G Protein Gamma Subunits Is Important for Their Function as Governors of G Protein Signaling

Emerging evidence suggests that heterotrimeric G protein gamma subunits (Gγ) are important governors of G protein signaling, a function that is mediated through GPCR‐ and pH‐dependent combinatorial phosphorylation of their intrinsically disordered N‐terminal tails (Gγ‐Nt) that controls Gβγ/effector interactions and signaling. Intrinsic disorder is a universally conserved structural feature of all Gγ subunit N‐termini, which prompted us to hypothesize that, beyond phosphorylation, intrinsic disorder itself is inherently important to the signal‐governing roles of Gγ subunits. To test this hypothesis we devised a strategy in which single amino acid substitutions are sequentially introduced into the Gγ tail, producing a series of isoforms that proceed step‐wise from a fully‐disordered to fully‐ordered (α‐helical) Nt tail structure. As a control for the increasing mutation load, we compare these mutants to those in which the same number of amino acid substitutions are incorporated that do not alter the inherent structural disorder of the tail. These mutant isoforms were then structurally analyzed by circular dichroism (CD) in vitro, by molecular dynamics (MD) simulation in silico, and by functional analysis of Gβγ‐dependent molecular signaling in vivo. Here, we apply this approach to the yeast Gγ subunit, Ste18. CD and MD analyses of Ste18‐Nt tail isoforms indicate that a successful transition from a fully‐disordered to fully‐ordered state is achievable through precise point mutation. Replacing the wild type Gγ subunit with each of the mutant isoforms in yeast, we further show that intrinsic disorder of Gγ‐Nt controls the stability of the Gγ subunit in a manner that is proportional to the loss of intrinsic disorder in vivo. pH‐dependent phosphorylation at Ser3 in the tail is largely unaffected by these changes. However, unexpectedly, we found that the GPCR‐dependent phosphorylation site, Ser7, becomes pH‐sensitive in response to changes in tail structure. Ongoing experiments reveal the effects of Ste18‐Nt tail structure on the interaction of yeast Gbg with its primary effector Ste5, and subsequent effects on activation of MAPKs, which have been shown to be highly sensitive to Gγ‐Nt tail phosphorylation previously. Taken together, these data provide evidence that intrinsic structural disorder plays a direct role in functionality of Gγ subunits as governors of G proteins signaling and substantiates the rationale for exploring similar roles for these tails in mammalian Gβγ‐dependent signaling pathways.

Simultaneous Improvement of Grain Yield and Quality through Manipulating Two Type C G Protein Gamma Subunits in Rice.

Heterotrimeric G protein signaling is an evolutionarily conserved mechanism in diverse organisms that mediates intracellular responses to external stimuli. In rice, the G proteins are involved in the regulation of multiple important agronomic traits. In this paper, we present our finding that two type C G protein gamma subunits, DEP1 and GS3, antagonistically regulated grain yield and grain quality. The DEP1 gene editing we conducted, significantly increased the grain number per panicle but had a negative impact on taste value, texture properties, and chalkiness-related traits. The GS3 gene editing decreased grain number per panicle but significantly increased grain length. In addition, the GS3 gene-edited plants showed improved taste value, appearance, texture properties, and Rapid Visco Analyser (RVA) profiles. To combine the advantages of both gs3 and dep1, we conducted a molecular design breeding at the GS3 locus of a “super rice” variety, SN265, which has a truncated dep1 allele with erect panicle architecture, high-yield performance, and which is of mediocre eating quality. The elongated grain size of the sn265/gs3 gene-edited plants further increased the grain yield. More importantly, the texture properties and RVA profiles were significantly improved, and the taste quality was enhanced. Beyond showcasing the combined function of dep1 and gs3, this paper presents a strategy for the simultaneous improvement of rice grain yield and quality through manipulating two type C G protein gamma subunits in rice.

Changes in long-chain fatty acid composition of milk fat globule membrane and expression of mammary lipogenic genes in dairy cows fed sunflower seeds and rumen-protected choline.

The aim of this experiment was to investigate the effect of dietary supplementation of crushed high oleic sunflower seeds (HOS) and rumen-protected choline (RPC) on the fatty acid (FA) profile of phospholipids and sphingomyelin and mammary transcription of genes that are important for milk fat synthesis and de novo synthesis of sphingolipids. Twenty-four cows were divided into four groups that either received an unsupplemented diet (Control), the Control diet supplemented with 50g RPC per day, a diet supplemented with HOS at 10% of dry matter, or RPC and HOS in combination (RPC+HOS). RPC supplementation had no effect on the FA composition of milk or sphingomyelin. Cows receiving RPC and RPC+HOS had increased incorporation of C22:5 (n-3) into phospholipids. Milk FA proportion of C18:0 and C18:1 isomers was increased in cows receiving HOS (HOS and RPC+HOS). Sphingomyelin proportion of C22:0 was increased in cows receiving HOS and RPC+HOS, at the expense of C23:0. HOS supplementation further increased the proportion of unsaturated fatty acids (UFA) in milk phospholipids. HOS supplementation increased mammary transcription of UDP-glucose ceramide glycosyltransferase (UGCG), sterol response element-binding protein cleavage-activating protein (SCAP) and peroxisome proliferation-activated receptor Gamma subunit C 1b (PPARGC1b), and reduced transcription of insulin induced gene 1 (INSIG1) and fatty acid-binding protein 3 (FABP3). Dietary supplementation of RPC increased mammary transcription of fatty acid desaturase 1 (FADS1) and longevity assurance gene 2 (LASS2), and reduced transcription of sphingomyelin synthase (SGMS). The results show that the FA profile of milk phospholipids is sensitive to dietary lipid supplementation and, to a minor degree, RPC supplementation. Furthermore, transcription of genes that are important for milk fat synthesis and sphingolipids synthesis is affected by dietary supplementation of RPC and HOS.

G Protein Gamma Subunits: Phosphorylation‐Mediated Governors of G protein Signaling in Humans?

Intracellular heterotrimeric G proteins (consisting of Gα, Gβ, and Gγ subunits), interface with 7‐transmembrane G protein coupled receptors (GPCRs) and function as the primary transducers of extracellular signals such as hormones, neurotransmitters, as well as pharmaceutical compounds. Upon binding their cognate ligands, GPCRs promote the dissociation of Gα and Gβγ subunits, which are then free to interact with effector proteins that promote intracellular signaling responses to the stimulus. Within this context, Gγ subunits are thought of primarily as membrane anchors for Gβ subunits, which must reside at the plasma membrane to facilitate functional protein interactions. However, emerging evidence suggests that Gγ subunits serve as phosphorylation‐dependent governors of G protein signaling. We have recently demonstrated this in the budding yeast model system, in which negative feedback phosphorylation of the intrinsically disordered N‐terminal tail of Gγ/Ste18 disrupts Gβγ/effector interactions and inhibits the normal GPCR‐dependent activation of MAPKs (Choudhury S. et al, Cell Rep. 2018). More recently, we have discovered that the yeast Gγ subunit undergoes combinatorial phosphorylation in response to several different forms of extracellular stimuli, including osmotic stress, pH stress, glucose deprivation, and cell cycle progression, in addition to GPCR activation. In light of these discoveries, we have hypothesized that Gγ phosphorylation plays a similar role in mammalian GPCR pathways. Indeed, we show that, like Gγ/Ste18, all mammalian Gγ subunits have an intrinsically disordered N‐terminus and most also undergo multi‐site phosphorylation. Here we provide new evidence to evaluate the hypothesis that Gγ phosphorylation is a governor of G protein signaling in mammals by demonstrating that phosphorylation is readily detectable on many different human Gγ subunits (Gγ3, 4, 5, 7, 10, 12) and by systematically testing the requirement for phosphorylation in proper Gβγ‐specific signaling pathways including the activation of PKD1 kinase by phospholipase C β2/3.Support or Funding InformationNIH

The G Protein Gamma Subunit, Ste18, is a Phosphorylation‐Dependent pH Sensor that Controls MAPK Activation in Yeast

As essential transducers of extracellular stimuli such as hormones and neurotransmitters, G protein signaling (GPS) systems – comprised of seven transmembrane receptors (GPCRs), heterotrimeric G‐proteins (Gαβγ), and regulators of G‐protein signaling (RGS) proteins – are important regulators of cell/environment homeostasis in all eukaryotes. Yeast harbor a single canonical GPS system that regulates a pheromone‐dependent mating response, which has long served as a tool for discovering regulators fundamental for all eukaryotic GPS systems. In wild type yeast, the activation rate and amplitude of pheromone signaling is severely dampened by negative feedback phosphorylation of the canonical Gγ subunit, Ste18, and the MAPK scaffold, Ste5. Here, we show that Ste18 is a pH sensor that undergoes combinatorial phosphorylation/dephosphorylation in response to fluctuations in intracellular pH (pHi). Using a site‐specific phosphorylation‐dependent electrophoretic mobility assay and a pHi probe (pHlourin), we show that Ste18S3 is rapidly phosphorylated in response to intracellular acidification, while Ste18S7 is rapidly phosphorylated in response to mating pheromone. When pHi is lowered, basal phosphorylation on Ste18S7 decreases concomitantly with increasing phosphorylation on Ste18S3. In contrast, pheromone stimulation activates phosphorylation on Ste18S7, but not Ste18S3. We show that phosphomimic mutations on either phosphosite function to prohibit phosphorylation of the other non‐mutated site, demonstrating that phosphorylation is mutually exclusive. Furthermore, phosphorylation at either site, mimicked by glutamate mutations, is sufficient to inhibit ultra‐fast and intense output from the pheromone pathway. Thus, our findings suggest that Ste18Nt acts as a phosphorylation‐dependent pH‐sensor that is capable of restricting pheromone pathway output under acid stress conditions when yeast mating is sub‐optimal. Considering that dysregulation of pHi is linked to chronic and life‐threatening diseases such as cancer, stroke, and Alzheimer's disease, our data reveal the possibility that Gγ subunits serve as pH sensors important for regulating G protein signaling in such diseases.Support or Funding InformationNational Institutes of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Farnesylation of the Transducin G Protein Gamma Subunit Is a Prerequisite for Its Ciliary Targeting in Rod Photoreceptors.

Primary cilia are microtubule-based organelles, which protrude from the plasma membrane and receive a wide range of extracellular signals. Various cilia use G protein-coupled receptors (GPCRs) for the detection of these signals. For instance, vertebrate rod photoreceptors use their cilia (also called outer segments) as antennae detecting photons by GPCR rhodopsin. Rhodopsin recognizes incoming light and activates its G protein, transducin, which is composed of three subunits α, β, and γ. Similar to all G protein γ subunits, the transducin Gγ1 subunit undergoes C-terminal prenylation resulting in the addition of an isoprenoid farnesyl; however, the significance of this posttranslational modification is unclear. To study the role of the farnesyl group, we genetically introduced a mutant Gγ1 that lacked the prenylation site into the retinal photoreceptors of mice. The biochemical and physiological analyses of these mice revealed that mutant Gγ1 dimerizes with the endogenous transducin Gβ1 subunit and that the resulting Gβγ dimers display reduced hydrophobicity. Although mutant Gβγ dimers could form a heterotrimeric G protein, they could not mediate phototransduction. This deficiency was due to a strong exclusion of non-farnesylated Gβγ complexes from the cilia (rod outer segments). Our results provide the first evidence that farnesylation is required for trafficking of G-protein βγ subunits to the cilium of rod photoreceptors.

Abstract LB-B07: Photoreceptor phosphodiesterase 6H expression regulates HCT116 proliferation and cell cycle progression

Abstract Phosphodiesterase 6 (PDE6) protein complex, which is responsible for the turnover of the second messenger cyclic guanosine monophosphate (cGMP) and is involved in visual signal transduction of light stimuli, is highly expressed in certain cancer cell lines. Despite having been discovered in the outer segment of rods and cones of the retina, PDE6 protein is also detected in brain, adrenal glands, lungs, and gonads. Genes encoding the catalytic subunits of the enzyme are known to be upregulated in certain breast, non-small cell lung cancer and medulloblastoma cells. PDE6H, which encodes the cone-specific inhibitory gamma subunit of PDE6 protein, is part of the gene signature of achromatopsia and congenital stationary night blindness while its relation to cancer remains unclear. We recently identified PDE6H as a cell cycle controller in HCT116 colorectal cancer cell line, as part of an siRNA screen in which DNA content measurement via flow cytometry was undertaken to monitor cell cycle distribution. The screen was conducted with a custom library of metabolic genes commonly amplified in primary tumours, as well as those known to be involved in the key regulatory points of metabolic pathways. We have shown that PDE6H knockdown and pharmacological inhibition of PDE6 protein hindered the activity of the protein complex, causing an accumulation of cGMP in various colorectal and breast cancer cell lines. This inhibition resulted in a G1 cell cycle arrest and disruption of G1/S checkpoint mechanism. PDE6H knockdown inhibited cell proliferation and cellular oxidative phosphorylation. It also altered mitochondrial morphology and induced apoptosis. Our results suggest that the gamma subunit is important for the function of photoreceptor phosphodiesterase PDE6 complex and PDE6H inhibition can be explored as an anti-proliferative strategy. Citation Format: Ceren Yalaz, Syed Haider, Jianzhou Chen, Remko Prevo, Mick Woodcock, Adrian L. Harris, Ruth J. Muschel. Photoreceptor phosphodiesterase 6H expression regulates HCT116 proliferation and cell cycle progression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B07.

Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit

Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.

Protein Geranylgeranyltransferase Type 1 as a Target in Cancer.

The process of protein prenylation involves the covalent linkage of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoid lipds to conserved cysteine residues in the carboxyl-terminus of proteins. Protein geranylgeranyltransferase I (GGTase-I) is the enzyme that catalyzes the addition of the geranylgeranyl moiety from geranylgeranyl pyrophosphate to the target protein, which contains a Cterminal consensus sequence termed a CaaX motif. Geranylgeranylation is important to the function of a number of proteins, including the majority of Rho GTPases, G protein gamma subunits, and several other regulatory proteins. Studies over the past two decades have revealed that many of these proteins contribute to tumor development and metastasis. Blocking Rho GTPase activity through inhibition of GGTase-I in particular has been advanced as a potential strategy for disease therapy. This review will provide an overview of the CaaX prenyltransferases, the rationale for targeting GGTase-I in cancer in particular, and the current status of GGTase-I inhibitor (GGTI) development.

Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.

Functional analyses and affinity-alteration of receptors and enzymes based on membrane recruitment of yeast guanine nucleotide-binding protein gamma subunit

To investigate fundamental processes conserved in all eukaryotic cells, the budding yeast Saccharomyces cerevisiae has being widely used as a model organism. In particular, the yeast two-hybrid system is a powerful technique for analyzing protein–protein interactions and protein function in living cells. Here, we describe several approaches for investigating and regulating the activity of target proteins using the yeast guanine nucleotide-binding protein (G-protein) signaling machinery as the readout. These approaches are rapid and easy-to-use tools that support the design of regulatory factors against receptors, enzymes, and other proteins that have been identified as potential drug target molecules.

Central and C-terminal domains of heterotrimeric G protein gamma subunits differentially influence the signaling necessary for primordial germ cell migration

Heterotrimeric G protein signaling is involved in many pathways essential to development including those controlling cell migration, proliferation, differentiation and apoptosis. One key developmental event known to rely on proper heterotrimeric G protein signaling is primordial germ cell (PGC) migration. We previously developed an in vivo PGC migration assay that identified differences in the signaling capacity of G protein gamma subunits. In this study we developed Gγ subunit chimeras to determine the regions of Gγ isoforms that are responsible for these differences. The central section of the Gγ subunit was found to be necessary for the ability of a Gγ subunit to mediate signaling involved in PGC migration. Residues found in the carboxy-terminal segment of Gγ transducin ( gngt1) were found to be responsible for the ability of this subunit to disrupt PGC migration. The type of prenylation did not affect the ability of a Gγ subunit to reverse prenylation-deficient-Gγ-induced PGC migration defects. However, a version of gng2, engineered to be farnesylated instead of geranylgeranylated, still lacks the ability to reverse PGC migration defects known to result from treatment of zebrafish with geranylgeranyl transferase inhibitors (GGTI), supporting the notion that Gγ subunits are one of several protein targets that need to be geranylgeranylated to orchestrate the proper long-range migration of PGCs.

Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit

Cellular senescence is a process wherein proliferating cells undergo permanent cell cycle arrest while remaining viable. Senescence results in enhanced secretion of proteins that promote cancer and inflammation. We report here that the structure of the Golgi complex which regulates secretion is altered in senescent cells. In cells where senescence is achieved by replicative exhaustion or in cells wherein senescence has been induced with BrdU treatment dependent stress, the Golgi complex is dispersed. The expression of a G protein γ subunit, γ11, capable of translocation from the plasma membrane to the Golgi complex on receptor activation increases with senescence. Knockdown of γ11 or overexpression of a dominant negative γ3 subunit inhibits Golgi dispersal induced by senescence. Overall these results suggest that in cellular senescence an upregulated G protein gamma subunit mediates alterations in the structure of the Golgi.

SAXS, CD and DLS studies on plant heterotrimeric G protein Gamma subunit; AGG2

SAXS, CD and DLS studies on plant heterotrimeric G protein Gamma subunit; AGG2

Prenylation-deficient heterotrimeric G protein gamma subunits reveal GPCR-mediated signaling events in vivo

Prenylation-deficient heterotrimeric G protein gamma subunits reveal GPCR-mediated signaling events in vivo

Prenylation-deficient G protein gamma subunits disrupt GPCR signaling in the zebrafish

Prenylation of G protein gamma (γ) subunits is necessary for the membrane localization of heterotrimeric G proteins and for functional heterotrimeric G protein coupled receptor (GPCR) signaling. To evaluate GPCR signaling pathways during development, we injected zebrafish embryos with mRNAs encoding Gγ subunits mutated so that they can no longer be prenylated. Low-level expression of these prenylation-deficient Gγ subunits driven either ubiquitously or specifically in the primordial germ cells (PGCs) disrupts GPCR signaling and manifests as a PGC migration defect. This disruption results in a reduction of calcium accumulation in the protrusions of migrating PGCs and a failure of PGCs to directionally migrate. When co-expressed with a prenylation-deficient Gγ, 8 of the 17 wildtype Gγ isoforms individually confer the ability to restore calcium accumulation and directional migration. These results suggest that while the Gγ subunits possess the ability to interact with G Beta (β) proteins, only a subset of wildtype Gγ proteins are stable within PGCs and can interact with key signaling components necessary for PGC migration. This in vivo study highlights the functional redundancy of these signaling components and demonstrates that prenylation-deficient Gγ subunits are an effective tool to investigate the roles of GPCR signaling events during vertebrate development.

Proteomic profiling of PrP27‐30‐enriched preparations extracted from the brain of hamsters with experimental scrapie

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation in the CNS of a pathological conformer (PrP(TSE)) of the host-encoded cellular prion protein (PrP(C)). PrP(TSE) has a central role in the pathogenesis of the disease but other factors are likely involved in the pathological process. In this work we employed a multi-step proteomic approach for the identification of proteins that co-purify with the protease-resistant core of PrP(TSE) (PrP27-30) extracted from brains of hamsters with experimental scrapie. We identified ferritin, calcium/calmodulin-dependent protein kinase alpha type II, apolipoprotein E, and tubulin as the major components associated with PrP27-30 but also trace amounts of actin, cofilin, Hsp90alpha, the gamma subunit of the T-complex protein 1, glyceraldehyde 3-phosphate dehydrogenase, histones, and keratins. Whereas some of these proteins (tubulin and ferritin) are known to bind PrP, other proteins (calcium/calmodulin-dependent protein kinase alpha type II, Hsp90alpha) may associate with PrP(TSE) fibrils during disease. Apolipoprotein E and actin have been previously observed in association with PrP(TSE), whereas cofilin and actin were shown to form abnormal rods in the brain of patients with Alzheimer disease. The roles of these proteins in the development of brain lesions are still unclear and further work is needed to explain their involvement in the pathogenesis of TSEs.

Heterotrimeric G Proteins Regulate a Noncanonical Function of Septate Junction Proteins to Maintain Cardiac Integrity in Drosophila

The gene networks regulating heart morphology and cardiac integrity are largely unknown. We previously reported a role for the heterotrimeric G protein gamma subunit 1 (Ggamma1) in mediating cardial-pericardial cell adhesion in Drosophila. Here we show G-oalpha47A and Gbeta13F cooperate with Ggamma1 to maintain cardiac integrity. Cardial-pericardial cell adhesion also relies on the septate junction (SJ) proteins Neurexin-IV (Nrx-IV), Sinuous, Coracle, and Nervana2, which together function in a common pathway with Ggamma1. Furthermore, Ggamma1 signaling is required for proper SJ protein localization, and loss of at least one SJ protein, Nrx-IV, induces cardiac lumen collapse. These results are surprising because the embryonic heart lacks SJs and suggest that SJ proteins perform noncanonical functions to maintain cardiac integrity in Drosophila. Our findings unveil the components of a previously unrecognized network of genes that couple G protein signaling with structural constituents of the heart.

Somatostatin modulates voltage-dependent Ca2+ channels in GH3 cells via a specific G(o) splice variant.

In rat pituitary GH3 cells Ca2+ current through L-type channels is reduced by somatostatin. This modulation of channel activity by somatostatin receptors is mediated by a guanine nucleotide-binding regulatory protein (G protein). It is sensitive to pertussis toxin, indicating the involvement of a G(o)- or Gi-type G protein in this pathway. The identity of this G protein was determined by suppressing the expression of endogenous G proteins individually via intranuclear injection of antisense oligonucleotides. This method was applied to GH3 cells to screen several G protein alpha, beta and gamma subunits for their roles in the defined signal transduction pathway. The loss of somatostatin's modulating activity on the voltage-dependent Ca2+ channel after oligonucleotide injection revealed the involvement of G(o) alpha 2 beta 1 gamma 3 to the exclusion of other closely related subtypes.