Expression pattern in the antennae of a newly isolated lepidopteran Gq protein alpha subunit cDNA.

From the antennae of the moth Mamestra brassicae, we have identified a lepidopteran G protein alpha subunit belonging to the Gq family, through immunological detection in crude antennal extract and antennal primary cell cultures, followed by molecular cloning. The complete cDNA sequence (1540 bp) contains an open reading frame encoding a protein of 353 amino acids. This deduced sequence possesses all of the characteristics of the Gq family and shares a very high degree of amino-acid sequence identity with vertebrate (80% with mouse or human Gqalpha) and invertebrate subunits (varying between 60 and 87% for Gqalpha from organisms as diverse as sponge and Drosophila). The expression pattern of the Gq subunit in adult antennae was associated with the olfactory sensilla suggesting a specific role in olfaction. These data provide molecular evidence for a component of the phosphoinositide signaling pathway in moth antennae: this G protein alpha subunit may be involved in the olfaction transduction process through interaction with G-protein-coupled receptors, stimulating the phospholipase C mediated second messenger pathway.     

Tissue development and RNA control: "HOW" is it coordinated?

The regulation of developmental processes at the RNA level enables selective and rapid modulation of gene expression. Studies in model organisms revealed the essential contribution of the signal transduction and activation of RNA (STAR) family of RNA binding proteins to developmental processes. STAR proteins coordinate the proper timing of developmental events by delaying expression or altering the mRNA or protein levels of essential genes. Recent functional analysis of the Drosophila melanogaster STAR protein, Held Out Wing (HOW), in the context of embryonic development, provided insight into its mode of activity. Here, we describe HOW's activity in the temporal repression or elevation of gene expression that is essential for coordinating the correct timing of instructive signals.     

Expression of Frizzled genes in the developing chick eye

Frizzleds are transmembrane receptors that can transduce signals dependent upon binding of Wnts, a large family of secreted glycoproteins homologous to the Drosophila wingless (wg) gene product and critical for a wide variety of normal and pathological developmental processes. In the nervous system, Wnts and Frizzleds play an important role in anterior-posterior patterning, cell fate decisions, proliferation, and synaptogenesis. However, little is known about the role of Frizzled signaling in the developing eye. We isolated cDNAs for ten chick Frizzleds and analyzed the spatial and temporal expression patterns during eye development in the chick embryo. Frizzled-1 to -9 are specifically expressed in the eye at various stages of development and show a complex and partially overlapping pattern of expression.     

Insect G protein-coupled receptors and signal transduction

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins (7-TM) that transduce extracellular signals into cellular physiological responses through the activation of heterotrimeric guanine nucleotide binding proteins (alpha beta gamma subunits). Their general properties are remarkably well conserved during evolution. Despite this general resemblance, a large variety of different signals are mediated via this category of receptors. Several GPCR-(sub)families have an ancient origin that is situated before the divergence of Protostomian and Deuterostomian animals. Nevertheless, an enormous diversification has occurred since then. The availability of novel sequence information is growing very rapidly as a result of molecular cloning experiments and of metazoan genome (Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens) and EST (expressed sequence tags) sequencing projects. The Drosophila Genome Sequencing Project will certainly have an important impact on insect signal transduction and receptor research. In parallel, convenient expression systems and functional assay procedures will be needed to investigate insect receptor properties and to monitor the effects of natural and artificial ligands. The study of the evolutionary aspects of G protein-coupled receptors and of their signaling pathways will probably reveal insect-specific features. More insight into these features may result in novel methods and practical applications. Arch.     

Folic acid stimulation of the Gα4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium

In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein alpha subunit encoded by the Galpha4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type Galpha4 gene, due to high copy gene dosage (Galpha4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, Galpha4HC prespore cell-specific gene expression and spore production were rescued by the presence of wild-type cells, indicating that prespore cell development in Galpha4HC cells is limited by the absence of an intercellular signal. Transplanted wild-type tips were sufficient to rescue Galpha4HC prespore cell development, suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, Galpha4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the Galpha4 subunit functions cell-autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild-type but not galpha4 null mutant aggregates, indicating that folic acid can inhibit cell-type-specific differentiation by stimulation of the Galpha4-mediated signal transduction pathway. The results of this study suggest that Galpha4-mediated signals can regulate cell-type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk-cell development.     

G13alpha-mediated PYK2 activation. PYK2 is a mediator of G13alpha -induced serum response element-dependent transcription

G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.     

Activation of nematode G protein GOA-1 by the human muscarinic acetylcholine receptor M2 subtype. Functional coupling of G-protein-coupled receptor and G protein originated from evolutionarily distant animals

Signal transduction mediated by heterotrimeric G proteins regulates a wide variety of physiological functions. We are interested in the manipulation of G-protein-mediating signal transduction using G-protein-coupled receptors, which are derived from evolutionarily distant organisms and recognize unique ligands. As a model, we tested the functionally coupling GOA-1, G alpha(i/o) ortholog in the nematode Caenorhabditis elegans, with the human muscarinic acetylcholine receptor M2 subtype (M2), which is one of the mammalian G alpha(i/o)-coupled receptors. GOA-1 and M2 were prepared as a fusion protein using a baculovirus expression system. The affinity of the fusion protein for GDP was decreased by addition of a muscarinic agonist, carbamylcholine and the guanosine 5'-[3-O-thio]triphosphate ([35S]GTPgammaS) binding was increased with an increase in the carbamylcholine concentrations in a dose-dependent manner. These effects evoked by carbamylcholine were completely abolished by a full antagonist, atropine. In addition, the affinity for carbamylcholine decreased under the presence of GTP as reported for M2-G alpha(i/o) coupling. These results indicate that the M2 activates GOA-1 as well as G alpha(i/o).     

New thoughts on the role of the beta-gamma subunit in G-protein signal transduction.

Heterotrimeric G proteins are involved in numerous biological processes, where they mediate signal transduction from agonist-bound G-protein-coupled receptors to a variety of intracellular effector molecules and ion channels. G proteins consist of two signaling moieties: a GTP-bound alpha subunit and a beta-gamma heterodimer. The beta-gamma dimer, recently credited as a significant modulator of G-protein-mediated cellular responses, is postulated to be a major determinant of signaling fidelity between G-protein-coupled receptors and downstream effectors. In this review we have focused on the role of beta-gamma signaling and have included examples to demonstrate the heterogeneity in the heterodimer composition and its implications in signaling fidelity. We also present an overview of some of the effectors regulated by beta-gamma and draw attention to the fact that, although G proteins and their associated receptors play an instrumental role in development, there is rather limited information on beta-gamma signaling in embryogenesis.     

The transduction signalling protein Go during embryonic development of Drosophila melanogaster

G proteins are heterotrimeric proteins that play a key role in signalling transduction conveying signals from cell surface receptors to intracellular effector proteins. In particulate preparations from Drosophila melanogaster embryos, only one substrate of 39,000-40,000 molecular weight could be ADP-ribosylated with pertussis toxin. This substrate reacted in immunoblotting and immunoprecipitation experiments with a polyclonal antibody directed against the carboxy-terminal sequence of the alpha subunit of the mammalian Go protein. The Drosophila Go alpha protein was present at all stages of embryonic development; however, its expression markedly increased after 10 h embryogenesis, a period of time during which there is an active development of axonal tracts. Immunolocalization on whole mount embryos has indicated that this protein is principally localized in the CNS and is mainly restricted to the neuropil without any labelling of the cell bodies. In contrast, all the axon tracts of the CNS appeared to be highly labelled. The distribution of the Go alpha protein was also examined in several neurogenic mutants. The Go alpha protein expression was not altered in any of them but the pattern of labelling was disorganized as was the neuronal network. These results suggest a possible role for the Go protein during axonogenesis.     

Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade

This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus, focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.     

G protein coupled growth factor receptor tyrosine kinase: no longer an oxymoron

Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are 2 such major signaling hubs in eukaryotes. Canonical signal transduction via trimeric G proteins is spatially and temporally restricted, i.e., triggered exclusively at the plasma membrane (PM) by agonist activation of G-protein-coupled receptors (GPCRs) via a process that completes within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a non-canonical pathway for activation of trimeric G proteins by the non-receptor GEF, GIV/Girdin, that has distinctive temporal and spatial features. Such activation can be triggered by multiple growth factor RTKs, can occur at the PM and on internal membranes discontinuous with the PM, and can continue for prolonged periods of time. The molecular mechanisms that govern such non-canonical G protein activation and the relevance of this new paradigm in health and disease is discussed.     

A possible role for caveolin as a signaling organizer in olfactory sensory membranes

Fast kinetics and sensitivity of olfactory signaling raise the question of whether the participating proteins may be associated in supramolecular transduction complexes. We found evidence that caveolin proteins could play an important role in organizing signaling elements in olfactory sensory neurons. Western blot analysis indicated that caveolins are highly enriched in olfactory sensory membranes, where they co-localize in detergent-insoluble complexes with key components of the signaling pathways. Furthermore, the results of immunoprecipitation experiments suggest that G proteins and effector enzyme form preassembled subcellular complexes with caveolins. Since anti-caveolin antibodies and synthetic peptides derived from the scaffolding domains of caveolin-1 and caveolin-2 effectively attenuated second messenger responses in sensory cilia preparations in a characteristic manner, the data led to the suggestion that caveolins could mediate the assembly of signaling complexes within specialized membrane microdomains of olfactory sensory neurons.     

Light-sensitive coupling of rhodopsin and melanopsin to G(i/o) and G(q) signal transduction in Caenorhabditis elegans

Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 of these receptors along with their cognate heterotrimeric G proteins. Here, we report properties of 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system of C. elegans with various genetically engineered alterations. Profound transient photoactivation of G(i/o) signaling by (b)isoRho led to a sudden and transient loss of worm motility dependent on cyclic adenosine monophosphate, whereas transient photoactivation of G(q) signaling by (h)Mo enhanced worm locomotion dependent on phospholipase Cβ. These transgenic C. elegans models provide a unique way to study the consequences of G(i/o) and G(q) signaling in vivo with temporal and spatial precision and, by analogy, their relationship to human neuromotor function.     

The promoter region of the Drosophila alpha 2-tubulin gene directs testicular and neural specific expression

The temporal and spatial expression pattern of the Drosophila melanogaster alpha 2-tubulin gene (alpha 2) has been investigated by examining the expression of an alpha 2-lacZ fusion gene. When this fusion gene is introduced into the germ line by P-element mediated transformation, expression is only detected in chordotonal organs and testes. Chordotonal organs, which are sensory organs of the peripheral nervous system, express the gene from late embryonic through adult stages in both males and females. Testicular expression occurs from larval through adult stages and is limited to germ-line cells, the primary and secondary spermatocytes and perhaps the early spermatids.     

Phosphorylation and nuclear translocation of a regulator of G protein signaling (RGS10).

Heterotrimeric G proteins are involved in the transduction of hormonal and sensory signals across plasma membranes of eukaryotic cells. Hence, they are a critical point of control for a variety of agents that modulate cellular function. Activation of these proteins is dependent on GTP binding to their alpha (Galpha) subunits. Regulators of G protein signaling (RGS) bind specifically to activated Galpha proteins, potentiating the intrinsic GTPase activity of the Galpha proteins and thus expediting the termination of Galpha signaling. Although there are several points in most G protein controlled signaling pathways that are affected by reversible covalent modification, little evidence has been shown addressing whether or not the functions of RGS proteins are themselves regulated by such modifications. We report in this study the acute functional regulation of RGS10 thru the specific and inducible phosphorylation of RGS10 protein at serine 168 by cAMP-dependent kinase A. This phosphorylation nullifies the RGS10 activity at the plasma membrane, which controls the G protein-dependent activation of the inwardly rectifying potassium channel. Surprisingly, the phosphorylation-mediated attenuation of RGS10 activity was not manifested in an alteration of its ability to accelerate GTPase activity of Galpha. Rather, the phosphorylation event correlates with translocation of RGS10 from the plasma membrane and cytosol into the nucleus.     

Calcium signal transduction from caveolae

Caveolae are specialized membrane microdomains that are found on the plasma membrane of most cells. Recent studies indicate that a variety of signaling molecules are highly organized in caveolae, where their interactions initiate specific signaling cascades. Molecules enriched in this membrane include G protein-coupled receptors, heterotrimeric GTP binding proteins, IP3 receptor-like protein, Ca2+ ATPase, eNOS, and several PKC isoforms. Direct measurements of calcium changes in endothelial cells suggest that caveolae may be sites that regulate intracellular Ca2+ concentration and Ca2+ dependent signal transduction. This review will focus on the role of caveolae in controlling the spatial and temporal pattern of intracellular Ca2+ signaling.     

Inhibition of the beta-catenin signaling pathway in blastocyst and uterus during the window of implantation in mice

Beta-catenin, the mammalian homolog of Drosophila armadillo protein, was first identified as a cadherin-associated protein at cell-cell junctions. Another function of beta-catenin is the transduction of cytosolic signals to the nucleus in a variety of cellular contexts, which usually are elicited by the active form of beta-catenin. The aim of the present study was to examine the potential role of active beta-catenin in the mouse embryo and uterus during embryo implantation. Active beta-catenin was detected differentially in mouse embryos and uteri during the peri-implantation period. Aberrant activation of beta-catenin by LiCl, a well-known glycogen synthase kinase-3 inhibitor, significantly inhibited blastocyst hatching and subsequent adhesion and outgrowth on fibronectin. Results obtained from pseudopregnant and implantation-delayed mice imply an important role for implanting blastocysts in the temporal and spatial changes of active beta-catenin in the uterus during the window of implantation. Collectively, these results suggest that the beta-catenin signaling pathway is inhibited in both blastocyst and uterus during the window of implantation, which may represent a new mechanism to synchronize the development of preimplantation embryos and differentiation of the uterus during this process.     

Cell-cell adhesion via the ECM: integrin genetics in fly and worm.

Integrins are essential for the development of the two genetically tractable invertebrate model organisms, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Just two integrins are present in C. elegans: one putative RGD binding integrin alphapat-2betapat-3, corresponding to Drosophila alphaPS2betaPS and vertebrate alpha5beta1, alphaVbeta1 and alpha8beta1, and one putative laminin binding integrin alphaina-1betapat-3, corresponding to Drosophila alphaPS1betaPS and vertebrate alpha3beta1, alpha6beta1 and alpha7beta1. In this review, the function of this minimal set of integrins during the development of these two invertebrates is compared. Despite the differences in bodyplan and developmental strategy, integrin adhesion to the extracellular matrix is required for similar processes: the formation of the link that translates muscle contraction into movement of the exoskeleton, cell migration, and morphogenetic interactions between epithelia. Other integrin functions, such as regulation of gene expression, have not yet been experimentally demonstrated in both organisms. Additional proteins have been characterised in each organism that are essential for integrin function, including extracellular matrix ligands and intracellular interacting proteins, but so far different proteins have been found in the two organisms. This in part represents the fact that the characterisation of the full set of interacting proteins is not complete in either system. However, in other cases different proteins appear to be used for similar functions in the two animals. The continued use of genetic approaches to identify proteins required for integrin function in these two model organisms should lead to the identification of the minimal set of conserved components that form integrin adhesive structures.     

Hyaluronan and CD44: strategic players for cell-matrix interactions during chondrogenesis and matrix assembly

Embryonic induction, soluble and insoluble factors, receptors, and signal transduction are orchestrated for the morphogenesis of the cartilage elements. The interaction of cells with the extracellular matrix (ECM) may lead to altered cellular response to morphogens based on the formation of new adhesive contacts, or the uncoupling of cell-matrix interactions. Hyaluronan's influence on cell behavior, and its intimate association with cells are accomplished by a wide variety of specific binding proteins for hyaluronan. The temporal expression of the hyaluronan receptor CD44 (which is expressed as several alternatively spliced variants) may be strategic to many of these cell-matrix interactions during chondrogenesis. CD44 expression is temporally coincident with the reduction of intercellular spaces at the regions of future cartilage deposition. The spatial organization of CD44 at the cell surface may function to establish or regulate the structure of the pericellular matrix dependent on a hyaluronan scaffold. As the ECM is modified during embryogenesis, the cellular response to inductive signals may be altered. An uncoupling of chondrocyte-hyaluronan interaction leads to chondrocytic chondrolysis. Thus, consideration of cell-matrix interactions during chondrogenesis, in the light of our current understanding of the temporal and spatial expression of signaling morphogens, should become a promising focus of future research endeavors.