AGS proteins, GPR motifs and the signals processed by heterotrimeric G proteins

A long term objective of our research effort is to define factors that influence the specificity and efficiency of signal propagation by heterotrimeric G-proteins (G). G-proteins play a central role in cellular communication mediating the cell response to numerous hormones and neurotransmitters. A major determinant of signalling specificity for heterotrimeric G-proteins is the cell specific expression of the subtypes of the primary signalling entities, receptor, G and effector (E). Another major site for regulating signalling specificity lies at the R-G or G-E interface where these interactions are influenced by cell architecture, the stoichiometry of signalling components and accessory proteins that may segregate the receptor to microdomains of the cell, regulate the efficiency and/or specificity of signal transfer and/or influence the activation state of G-protein independent of a classical G-protein coupled receptor. One strategy to address these issues in our laboratory involves the identification of cellular proteins that regulate the transfer of signal from receptor to G or directly influence the activation state of G independent of a classical G-protein coupled receptor. We identified three proteins, AGS1, AGS2 and AGS3 (for Activators of G-protein Signaling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1, 2 and 3 interact with different subunits and/or conformations of heterotrimeric G-proteins, selectively activate different G-proteins, provide unexpected mechanisms for regulation of the G-protein activation cycle and have opened up a new area of research related to the cellular role of G-proteins as signal transducers.     

Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein activator

A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 G-protein activator (NG-GPA)) increased [(35)S]guanosine 5'-O-(thiotriphosphate) ([(35)S]GTPgammaS) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Galpha(o). The NG-GPA also increased GTPgammaS binding to purified, recombinant Galpha(i2), Galpha(i3), and Galpha(o), but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgammaS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT(1)-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT(1)-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgammaS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G(i)/G(o) proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G(i)/G(o) signaling systems.     

Nrg1/ErbB signaling networks in Schwann cell development and myelination

Neuregulin-1 (Nrg1) provides a key axonal signal that regulates Schwann cell proliferation, migration and myelination through binding to ErbB2/3 receptors. The analysis of a number of genetic models has unmasked fundamental mechanisms underlying the specificity of the Nrg1/ErbB signaling axis. Differential expression of Nrg1 isoforms, Nrg1 processing, and ErbB receptor localization and trafficking represent important regulatory themes in the control of Nrg1/ErbB function. Nrg1 binding to ErbB2/3 receptors results in the activation of intracellular signal transduction pathways that initiate changes in Schwann cell behavior. Here, we review data that has defined the role of key Nrg1/ErbB signaling components like Shp2, ERK1/2, FAK, Rac1/Cdc42 and calcineurin in development of the Schwann cell lineage in vivo. Many of these regulators receive converging signals from other cues that are provided by Notch, integrin or G-protein coupled receptors. Signaling by multiple extracellular factors may act as key modifiers and allow Schwann cells at different developmental stages to respond in distinct manners to the Nrg1/ErbB signal.     

A Monte Carlo study of the dynamics of G-protein activation.

To link quantitatively the cell surface binding of ligand to receptor with the production of cellular responses, it may be necessary to explore early events in signal transduction such as G-protein activation. Two different model frameworks relating receptor/ligand binding to G-protein activation are examined. In the first framework, a simple ordinary differential equation model is used to describe receptor/ligand binding and G-protein activation. In the second framework, the events leading to G-protein activation are simulated using a dynamic Monte Carlo model. In both models, reactions between ligand-bound receptors and G-proteins are assumed to be diffusion-limited. The Monte Carlo model predicts two regimes of G-protein activation, depending upon whether the lifetime of a receptor/ligand complex is long or short compared with the time needed for diffusional encounters of complexes and G-proteins. When the lifetime of a complex is relatively short compared with the diffusion time, the movement of ligand among free receptors by binding and unbinding ("switching") significantly enhances G-protein activation. Receptor antagonists dramatically reduce G-protein activation and, thus, signal transduction in this case, and significant clustering of active G-proteins near receptor/ligand complexes results. The simple ordinary differential equation model poorly predicts G-protein activation for this situation. In the alternative case, when diffusion is relatively fast, ligand movement among receptors is less important and the simple ordinary differential equation model and Monte Carlo model results are similar. In this case, there is little clustering of active G-proteins near receptor/ligand complexes. Results also indicate that as the GTPase activity of the alpha-subunit decreases, the steady-state level of alpha-GTP increases, although temporal sensitivity is compromised.     

Timing is everything the role of kinetics in G protein activation

The binding of a drug to a G-protein coupled receptor initiates a complex series of dynamic events that ultimately leads to a cellular response. In addition to the concentrations of receptor, drug and G-protein, important determinants of the cellular response are the rates at which these species interact. However, most models for G-protein coupled receptor signaling are equilibrium models that neglect the role of reaction kinetics. A kinetic ternary-complex model of signaling through G-protein coupled receptors is presented. We demonstrate that this kinetic model can make significantly different predictions than an equilibrium ternary complex model, which provides a different perspective on multiple aspects of the signal transduction cascade, such as agonist efficacy, the effect of precoupled receptors, and the role of RGS proteins. Incorporation of the reaction kinetics is critical for a complete understanding of signal transduction and will ultimately impact the fields of drug discovery and drug design.     

Selectivity, cooperativity, and reciprocity in the interactions between the delta-opioid receptor, its ligands, and G-proteins

A better understanding of signal transduction mechanisms is of critical importance. Methodologies that allow studies to be done while receptors are incorporated into lipid bilayers are advantageous. One such technique is plasmon-waveguide resonance (PWR) spectroscopy, which can follow changes in conformation accompanying protein-ligand, protein-protein, and protein-lipid interactions occurring in G-protein-coupled receptors in real time with high sensitivity and without the need for molecular labeling. Here we investigated several aspects of human delta-opioid receptor (hDOR)-G-protein interactions: 1) the effect of different types of agonists on the interaction with individual G-protein subtypes; 2) the affinities of the separate G-protein alpha and betagamma subunits to different ligand-occupied states of the receptor; and 3) the effect of the presence of the G-protein on the interactions of the ligand with the receptor. To accomplish this we have incorporated the receptor into a solid supported lipid bilayer in the presence of ligand or G-protein and monitored the PWR spectral changes induced by the reciprocal G-protein or ligand interactions. We found a high degree of selectivity in the interactions of different agonist-bound states of the receptor with the different G-protein subtypes. This has important implications for agonist-directed trafficking and selective drug design. Studies with the separated alpha and betagamma subunits show that cooperativity exists in these interactions. The high affinities of the separated subunits to the receptor point to the possibility of independent promotion of specific signaling events. The presence of G-proteins increased the affinity of agonists to the hDOR, and caused faster binding kinetics and different ligand-induced conformational changes. Because ligand also influences G-protein binding, reciprocity exists between these two binding processes.     

Follicle-stimulating hormone increases guanine nucleotide-binding regulatory protein subunit alpha i-3 mRNA but decreases alpha i-1 and alpha i-2 mRNA in Sertoli cells.

FSH interacts with a guanine nucleotide-binding protein (G-protein)-coupled receptor, which in turn modulates signal transduction via the G-protein subunit alpha s. However, it is unknown whether FSH regulates alpha-subunit gene expression and whether G-protein alpha-subunit genes other than alpha s are modulated in FSH-stimulated signal transduction. Regulation of mRNA for alpha s and alpha i-2 was studied in primary cultures of rat Sertoli cells because these proteins are linked to the control of adenylyl cyclase. In addition, mRNA for alpha i-1 and alpha i-3 were quantified because these proteins are putatively linked to ion channels but have not been well characterized in the Sertoli cell. Northern blot analyses demonstrated that FSH induced a dose-dependent increase in steady state levels of alpha i-3 mRNA. In contrast, FSH caused a dose-dependent decrease in levels of alpha i-1 and alpha i-2 mRNA. No significant effect of FSH on alpha s mRNA levels was detectable. The time course of FSH effects showed a 75% decrease in alpha i-1 mRNA levels, a 50% decrease in alpha i-2 mRNA levels and a nearly 3-fold increase in levels of alpha i-3 mRNA between 4-6 h of treatment with 100 ng/ml FSH. Steady state levels of alpha i-1 and alpha i-2 mRNA returned to pretreatment levels after 10 h FSH treatment, while alpha i-3 mRNA returned to a new steady state level approximately 50% greater than the pretreatment level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor binding oscillations]

A kinetic analysis of the delta-opiate receptor agonist, [3H] DADLE, binding to cell (NG108-15) suspensions has led to the discovery of a new periodic biological phenomenon, namely, receptor activity oscillations. The absence of oscillations for the antagonist binding to the receptors suggests that oscillations are generated only as a result of receptor signal transformation. The correlation between the quantitative characteristics of the kinetic curves and the experimental conditions points to the fact that receptor binding oscillations may be due either to the receptor binding to the G-protein or the interaction of the receptor (or G-protein) with microtubules.     

Conversion of mechanical force into biochemical signaling

Physical forces play important roles in regulating cell proliferation, differentiation, and death by activating intracellular signal transduction pathways. How cells sense mechanical stimulation, however, is largely unknown. Most studies focus on cellular membrane proteins such as ion channels, integrins, and receptors for growth factors as mechanosensory units. Here we show that mechanical stretch-induced c-Src protein tyrosine kinase activation is mediated through the actin filament-associated protein (AFAP). Distributed along the actin filaments, AFAP can directly active c-Src through binding to its Src homology 3 and/or 2 domains. Mutations at these specific binding sites on AFAP blocked mechanical stretch-induced c-Src activation. Therefore, mechanical force can be transmitted along the cytoskeleton, and interaction between cytoskeletal associated proteins and enzymes related to signal transduction may convert physical forces into biochemical reactions. Cytoskeleton deformation-induced protein-protein interaction via specific binding sites may represent a novel intracellular mechanism for cells to sense mechanical stimulation.     

Membrane-initiated actions of thyroid hormones on the male reproductive system

The presence of specific nuclear receptors to thyroid hormones, described in prepubertal Sertoli cells, implies the existence of an early and critical influence of these hormones on testis development. Although the mechanism of action thyroid hormones has been classically established as a genomic action regulating testis development, our research group has demonstrated that these hormones exert several effects in Sertoli cells lacking nuclear receptor activation. These findings led to the identification of non-classical thyroid hormone binding elements in the plasma membrane of testicular cells. Through binding to these sites, thyroid hormones could exert nongenomic effects, including those on ion fluxes at the plasma membrane, on signal transduction via kinase pathways, on amino acid accumulation, on modulation of extracellular nucleotide levels and on vimentin cytoskeleton. The evidence of the participation of different K(+), Ca(2+) and Cl(-) channels in the mechanism of action of thyroid hormones, characterizes the plasma membrane as an important microenvironment able to coordinate strategic signal transduction pathways in rat testis. The physiological responses of the Sertoli cells to hormones are dependent on continuous cross-talking of different signal transduction pathways. Apparently, the choice of the signaling pathways to be activated after the interaction of the hormone with cell surface binding sites is directly related to the physiological action to be accomplished. Yet, the enormous complexity of the nongenomic actions of thyroid hormones implies that different specific binding sites located on the plasma membrane or in the cytosol are believed to initiate specific cell responses.     

Absolute macrophage dependency of T lymphocyte activation by mitogens.

A T lymphocyte subpopulation that contains only 0.3% macrophages and less than 2% B lymphocytes has been prepared from guinea pig lymph node cells by the use of two different types of adherence columns. This subpopulation does not porliferate in response to the mitogens Con A or PHA unless additional macrophages are added. The means by which macrophages restore T cell responsiveness to PHA has been investigated. Marcophages appear to function via two different distinct mechanisms in this experimental situation. The first mechanism involves the binding of PHA to the macrophage followed by the "presentation" of the mitogen to the T lymphocyte in a manner that induces cell activation. This presentation function requires that the macrophage be viable and metabolically active. The second mechanism by which macrophages function is by the elaboration of a soluble factor or factors. The presence of these factors has been reliably and reproducibly demonstrated by using a double-chambered, Marbrook-type tissue culture vessel. This soluble factor can induce activation of T lympohcytes with surface bound PHA in the apparent absence of any form of macrophage presentation. In contrast, the function of this factor is clearly distinct from that of the reducing agent, 2-mercaptoethanol, (2-ME) since 2-ME does not enable this T cell subpopulation to be activated by mitogens. On the basis of these observations, we propose that two distinct signals are required to activate this T lymphocyte subpopulation. One signal is delivered by the interaction of the mitogen with the T cell surface, and the second signal is delivered by a soluble factor(s) produced by macrophages. Whether all types of T lymphocytes require two signals to be activated, remains to be established.     

Kinases and G proteins join the Wnt receptor complex

Wnt proteins form a family of secreted signaling proteins that play a key role in various developmental events such as cell differentiation, cell migration, cell polarity and cell proliferation. It is currently thought that Wnt proteins activate at least three different signaling pathways by binding to seven transmembrane receptors of the Frizzled family and the co-receptor LRP6. Despite our growing knowledge of intracellular components that mediate a Wnt signal, the molecular events at the membrane have remained rather unclear. Now several publications(1-4) indicate that Frizzled receptors are G-protein coupled and kinases were identified that phosphorylate the co-receptor LRP6. These data deepen our understanding of Wnt-mediated signal transduction and provide more insight into how specificity may be achieved.     

New roles for Galpha and RGS proteins: communication continues despite pulling sisters apart

Large G protein alpha subunits and their attendant regulators of G-protein signaling (RGS) proteins control both intercellular signaling and asymmetric cell divisions by distinct pathways. The classical pathway, found throughout higher eukaryotic organisms, mediates intercellular communication via hormone binding to G-protein-coupled receptors (GPCRs). Recent studies have led to the discovery of GPCR-independent activation of Galpha subunits by the guanine nucleotide exchange factor RIC-8 in both asymmetric cell division and synaptic vesicle priming in metazoan organisms. Protein-protein interactions and protein function in each pathway are driven through the cycle of GTP binding and hydrolysis by the Galpha subunit. This review builds a conceptual framework for understanding RIC-8-mediated pathways by comparison with the mechanism of classical G-protein activation and inhibition in GPCR signaling.     

Interactions of mechanotransduction pathways

Integrins may serve as mechanosensors in endothelial cells (ECs): shear stress causes integrin-Shc association, assembly of the signaling complex and then leads to JNK activation. Flow also mediates selective and cell-specific alterations in vascular cell G-protein expression that correlate with changes in cell-signalling, G-protein functionality and modulate Ca2+ concentration. In this study, we explored the cross-talks between EC membrane mechanosensors, such as integrins, ion channels, and G-proteins in shear stress-induced signal transduction by their specific inhibition. Confluent monolayer of bovine aortic endothelial cells (BAECs) were incubated with or without specific inhibitors prior to shearing experiments. Our results showed an attenuation of integrin-Shc association under shear stress with RGD, and with PTX, but not with BAPTA/AM. The inhibitions of shear-activated JNK are similar for RGD and PTX. However, unlike for integrin association, the chelation of calcium reduced JNK activation. These results provide several lines of evidence of the interactions between different mechanosensors in ECs. First, integrin-Shc association required cell attachment and G-protein activity, but not intracellular calcium. Second, shear-induced JNK activation is regulated by multiple mechano-sensing mechanisms such as integrin, G-protein and calcium concentration.     

Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins

AGS3 (activator of G-protein signaling 3) was isolated in a yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins. As an initial approach to define the role of AGS3 in mammalian signal processing, we defined the AGS3 subdomains involved in G-protein interaction, its selectivity for G-proteins, and its influence on the activation state of G-protein. Immunoblot analysis with AGS3 antisera indicated expression in rat brain, the neuronal-like cell lines PC12 and NG108-15, as well as the smooth muscle cell line DDT(1)-MF2. Immunofluorescence studies and confocal imaging indicated that AGS3 was predominantly cytoplasmic and enriched in microdomains of the cell. AGS3 coimmunoprecipitated with Galpha(i3) from cell and tissue lysates, indicating that a subpopulation of AGS3 and Galpha(i) exist as a complex in the cell. The coimmunoprecipitation of AGS3 and Galpha(i) was dependent upon the conformation of Galpha(i3) (GDP GTPgammaS (guanosine 5'-3-O-(thio)triphosphate)). The regions of AGS3 that bound Galpha(i) were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro(463)-Ser(650)), each of which were capable of binding Galpha(i). AGS3-GPR domains selectively interacted with Galpha(i) in tissue and cell lysates and with purified Galpha(i)/Galpha(t). Subsequent experiments with purified Galpha(i2) and Galpha(i3) indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one Galpha(i) subunit at the same time. The AGS3-GPR domains effectively competed with Gbetagamma for binding to Galpha(t(GDP)) and blocked GTPgammaS binding to Galpha(i1). AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways.     

Human fetal skin fibroblast migration stimulated by the autocrine growth factor bFGF is mediated by phospholipase A(2) via arachidonic acid without the involvement of pertussis toxin-sensitive G-protein

We reported previously that human fetal skin fibroblast migration into a denuded area was stimulated by an autocrine factor, basic fibroblast growth factor (bFGF). Since the signal transduction pathway of this migration is unknown, we attempted to clarify it by comparing this fibroblast migration with a previously reported bovine endothelial cell migration into a wounded area stimulated by an addition of bFGF, in which the bFGF signal was mediated by phospholipase A(2)-coupled G-protein and phospholipase A(2) (PLA(2)) via arachidonic acid. Our study demonstrated that pertussis toxin, a specific inhibitor of PLA(2)-coupled G-protein, did not suppress human fetal skin fibroblast migration, but 2-(p-amylcinnamyl)amino-4-chlorobensoic acid (ONO-RS-082), a PLA(2) inhibitor, did. Since ONO-RS-082 is a non-specific PLA(2) inhibitor, a cytoplasmic, Ca-dependent PLA(2) (cPLA(2)) inhibitor, AACOCF3, was examined. AACOCF3 suppressed cell migration in certain concentrations. The PLA(2) inhibitor-suppressed cell migration was restored by adding arachidonic acid, and cell migration suppressed by anti-bFGF antibodies was restored by adding arachidonic acid. In addition, pertussis toxin did not suppress arachidonic acid release, which shows an action of PLA(2), but AACOCF3 did. These results indicate that human fetal skin fibroblast migration stimulated by an autocrine factor, bFGF, was mediated by PLA(2) via arachidonic acid without the involvement of PLA(2)-coupled G-protein.     

Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer

G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit interactions and the interaction of the G alpha subunit with RGS4. The assay uses a FRET pair consisting of a terbium cryptate chelate donor spectrally matched to an Alexa546 fluor acceptor, each of which is conjugated to separate protein binding partners, these being G alpha(i1):beta4gamma2 or G alpha(i1):RGS4. Under conditions favoring specific binding between labeled partners, high-affinity interactions were observed as a rapid increase (>fivefold) in the FRET signal. The specificity of these interactions was demonstrated using denaturing or competitive conditions which caused significant reductions in fluorescence (50-85%) indicating that labeled proteins were no longer in close proximity. We also report differential binding effects as a result of altered activation state of the G alpha(i1) protein. This assay confirms that interactions between G-protein subunits and RGS4 can be measured using TR-FRET in a cell- and receptor-free environment.     

Induced Effects of Sodium Ions on Dopaminergic G-Protein Coupled Receptors

G-protein coupled receptors, the largest family of proteins in the human genome, are involved in many complex signal transduction pathways, typically activated by orthosteric ligand binding and subject to allosteric modulation. Dopaminergic receptors, belonging to the class A family of G-protein coupled receptors, are known to be modulated by sodium ions from an allosteric binding site, although the details of sodium effects on the receptor have not yet been described. In an effort to understand these effects, we performed microsecond scale all-atom molecular dynamics simulations on the dopaminergic D₂ receptor, finding that sodium ions enter the receptor from the extracellular side and bind at a deep allosteric site (Asp2.50). Remarkably, the presence of a sodium ion at this allosteric site induces a conformational change of the rotamer toggle switch Trp6.48 which locks in a conformation identical to the one found in the partially inactive state of the crystallized human β₂ adrenergic receptor. This study provides detailed quantitative information about binding of sodium ions in the D₂ receptor and reports a possibly important sodium-induced conformational change for modulation of D₂ receptor function.