Signaling through G protein coupled receptors

Heterotrimeric G proteins (Gα, Gβ/Gγ subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane α-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Gα subunit. This leads to the dissociation of Gβ/Gγ dimer from Gα. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Gα-GTP is hydrolyzed to GDP and Gα becomes inactive (Gα-GDP), which leads to its re-association with the Gβ/Gγ dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.Key words: heterotrimeric G proteins, GPCRs, seven-transmembrane receptors, signal transduction, stress signaling     

When a G protein-coupled receptor does not couple to a G protein

Classically, G protein-coupled receptors (GPCRs) relay signals by directly activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Increasing evidence indicates that GPCRs may also signal through G protein-independent pathways. JAK/STATs, Src-family tyrosine kinases, GRKs/beta-arrestins, and PDZ domain-containing proteins have been suggested to directly relay signals from GPCRs independent of G proteins. In addition, our laboratory recently reported that the beta(2) adrenergic receptor (beta(2)AR) could switch from G protein-coupled to G protein-independent ERK (extracellular signal-regulated kinase) activation in an agonist dosage-dependent manner. This finding provides a novel mechanism for G protein-independent GPCR signaling. This review focuses on recent progress in understanding the mechanisms by which G protein-independent GPCR signaling occurs.     

GBF1 bears a novel phosphatidylinositol-phosphate binding module, BP3K, to link PI3Kγ activity with Arf1 activation involved in GPCR-mediated neutrophil chemotaxis and superoxide production

Most chemoattractants for neutrophils bind to the Gα(i) family of heterotrimeric G protein-coupled receptors (GPCRs) and release Gβγ subunits to activate chemotaxis and superoxide production. GIT2, a GTPase-activating protein for Arf1, forms a complex with Gβγ and is integral for directional sensing and suppression of superoxide production. Here we show that GBF1, a guanine nucleotide exchanging factor for Arf-GTPases, is primarily responsible for Arf1 activation upon GPCR stimulation and is important for neutrophil chemotaxis and superoxide production. We find that GBF1 bears a novel module, namely binding to products of phosphatidyl inositol 3-kinase (PI3K), which binds to products of PI3Kγ. Through this binding, GBF1 is translocated from the Golgi to the leading edge upon GPCR stimulation to activate Arf1 and recruit p22phox and GIT2 to the leading edge. Moreover, GBF1-mediated Arf1 activation is necessary to unify cell polarity during chemotaxis. Our results identify a novel mechanism that links PI3Kγ activity with chemotaxis and superoxide production in GPCR signaling.     

Backbone resonance assignments for G protein αi3 subunit in the GTP-bound state

Guanine-nucleotide binding proteins (G proteins) act as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of GDP-bound form of the G protein α subunit (Gα(GDP)) and G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα(GTP)) and Gβγ. Then, Gα(GTP) and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Each family transduces the signaling from different GPCRs to the specific effectors. Here, we established the backbone resonance assignments of human Gα_i3, a member of the i/o family, with a molecular weight of 41 K in complex with a GTP analogue, GTPγS.     

A Physiologically Required G Protein-coupled Receptor (GPCR)-Regulator of G Protein Signaling (RGS) Interaction That Compartmentalizes RGS Activity

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.     

Sensing and transduction of nutritional and chemical signals in filamentous fungi: Impact on cell development and secondary metabolites biosynthesis

Filamentous fungi respond to hundreds of nutritional, chemical and environmental signals that affect expression of primary metabolism and biosynthesis of secondary metabolites. These signals are sensed at the membrane level by G protein coupled receptors (GPCRs). GPCRs contain usually seven transmembrane domains, an external amino terminal fragment that interacts with the ligand, and an internal carboxy terminal end interacting with the intracellular G protein. There is a great variety of GPCRs in filamentous fungi involved in sensing of sugars, amino acids, cellulose, cell-wall components, sex pheromones, oxylipins, calcium ions and other ligands. Mechanisms of signal transduction at the membrane level by GPCRs are discussed, including the internalization and compartmentalisation of these sensor proteins. We have identified and analysed the GPCRs in the genome of Penicillium chrysogenum and compared them with GPCRs of several other filamentous fungi. We have found 66 GPCRs classified into 14 classes, depending on the ligand recognized by these proteins, including most previously proposed classes of GPCRs. We have found 66 putative GPCRs, representatives of twelve of the fourteen previously proposed classes of GPCRs, depending on the ligand recognized by these proteins. A staggering fortytwo putative members of the new GPCR class XIV, the so-called Pth11 sensors of cellulosic material as reported for Neurospora crassa and some other fungi, were identified. Several GPCRs sensing sex pheromones, known in yeast and in several fungi, were also identified in P. chrysogenum, confirming the recent unravelling of the hidden sexual capacity of this species. Other sensing mechanisms do not involve GPCRs, including the two-component systems (HKRR), the HOG signalling system and the PalH mediated pH transduction sensor. GPCR sensor proteins transmit their signals by interacting with intracellular heterotrimeric G proteins, that are well known in several fungi, including P. chrysogenum. These G proteins are inactive in the GDP containing heterotrimeric state, and become active by nucleotide exchange, allowing the separation of the heterotrimeric protein in active Gα and Gβγ dimer subunits. The conversion of GTP in GDP is mediated by the endogenous GTPase activity of the G proteins. Downstream of the ligand interaction, the activated Gα protein and also the Gβ/Gγ dimer, transduce the signals through at least three different cascades: adenylate cyclase/cAMP, MAPK kinase, and phospholipase C mediated pathways.     

A critical role of Gbetagamma in tumorigenesis and metastasis of breast cancer

A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Here we evaluated the role of Gβγ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking Gβγ signaling with Gα(t) or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of Gα(t) in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking Gβγ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that Gβγ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of Gβγ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of Gβγ signaling in promoting breast tumor growth and metastasis and suggest that targeting Gβγ may represent a novel therapeutic approach for breast cancer.     

WD40-repeat proteins control the flow of Gβγ signaling for directional cell migration

The ability of cells to generate a highly polarized intracellular signal through G protein-coupled receptors (GPCRs) is essential for their migration toward chemoattractants. The Gβγ subunits of heterotrimeric G proteins play a critical role in transmitting chemotactic signals from GPCRs via the activation of diverse effectors, including PLCβ and PI3K, primarily at the leading edge of cells. Although Gβγ can directly activate many of these effectors through protein-protein interactions in vitro, it remains unclear how Gβγ spatially and temporally orchestrates the activation of these effectors in vivo. A yeast two-hybrid screen for Gβ interacting proteins identified two WD40-repeat domain containing proteins, RACK1 and WDR26, which are predicted to serve as scaffolding/adaptor proteins. Previous data indicates that RACK1 negatively regulates Gβγ-mediated leukocyte migration by inhibiting Gβγ-stimulated PLCβ and PI3K activities. In contrast, recently published work by Sun et al. indicates that WDR26 promotes leukocyte migration by enhancing Gβγ-mediated signal transduction. These findings reveal a novel mechanism regulating Gβγ signaling during chemotaxis, namely through the positive and negative regulation of WDR26 and RACK1 on Gβγ to promote and fine tune Gβγ-mediated effector activation, ultimately governing the ability of cells to polarize and migrate toward a chemoattractant gradient.     

RhoA co-ordinates with heterotrimeric G proteins to regulate efficacy

Heterotrimeric G proteins have a critical role in mediating signal transduction by ligand-stimulated GPCRs. While activation of heterotrimeric G proteins is known to proceed via the G protein guanine nucleotide cycle, there is much uncertainty regarding the process that determines efficacy, the extent of response across signaling pathways. Gα_GTP can interact with multiple binding partners, including several effectors and regulators. Cross-talk by other receptor-signaling pathways can alter the response. It remains unclear whether G protein efficacy is regulated. This lack of clarity impairs our ability to predict and manipulate the pharmacological behavior of activated G proteins. This review will discuss emerging evidence that implicates monomeric RhoA in the process that regulates G_q efficacy.     

Non-canonical signaling and localizations of heterotrimeric G proteins

Heterotrimeric G proteins typically transduce signals from G protein-coupled receptors (GPCRs) to effector proteins. In the conventional G protein signaling paradigm, the G protein is located at the cytoplasmic surface of the plasma membrane, where, after activation by an agonist-bound GPCR, the GTP-bound Gα and free Gβγ bind to and regulate a number of well-studied effectors, including adenylyl cyclase, phospholipase Cβ, RhoGEFs and ion channels. However, research over the past decade or more has established that G proteins serve non-canonical roles in the cell, whereby they regulate novel effectors, undergo activation independently of a GPCR, and/or function at subcellular locations other than the plasma membrane. This review will highlight some of these non-canonical aspects of G protein signaling, focusing on direct interactions of G protein subunits with cytoskeletal and cell adhesion proteins, the role of G proteins in cell division, and G protein signaling at diverse organelles.     

Structural domains required for Caenorhabditis elegans G protein-coupled receptor kinase 2 (GRK-2) function in vivo

G protein-coupled receptor kinases (GRKs) are key regulators of signal transduction that specifically phosphorylate activated G protein-coupled receptors (GPCRs) to terminate signaling. Biochemical and crystallographic studies have provided great insight into mammalian GRK2/3 interactions and structure. However, despite extensive in vitro characterization, little is known about the in vivo contribution of these described GRK structural domains and interactions to proper GRK function in signal regulation. We took advantage of the disrupted chemosensory behavior characteristic of Caenorhabditis elegans grk-2 mutants to discern the interactions required for proper in vivo Ce-GRK-2 function. Informed by mammalian crystallographic and biochemical data, we introduced amino acid substitutions into the Ce-grk-2 coding sequence that are predicted to selectively disrupt GPCR phosphorylation, Gα(q/11) binding, Gβγ binding, or phospholipid binding. Changing the most amino-terminal residues, which have been shown in mammalian systems to be required specifically for GPCR phosphorylation but not phosphorylation of alternative substrates or recruitment to activated GPCRs, eliminated the ability of Ce-GRK-2 to restore chemosensory signaling. Disrupting interaction between the predicted Ce-GRK-2 amino-terminal α-helix and kinase domain, posited to stabilize GRKs in their active ATP- and GPCR-bound conformation, also eliminated Ce-GRK-2 chemosensory function. Finally, although changing residues within the RH domain, predicted to disrupt interaction with Gα(q/11), did not affect Ce-GRK-2 chemosensory function, disruption of the predicted PH domain-mediated interactions with Gβγ and phospholipids revealed that both contribute to Ce-GRK-2 function in vivo. Combined, we have demonstrated functional roles for broadly conserved GRK2/3 structural domains in the in vivo regulation of organismal behavior.     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

The G protein Gαs acts as a tumor suppressor in sonic hedgehog signaling-driven tumorigenesis

G protein-coupled receptors (GPCRs) are critical players in tumor growth and progression. The redundant roles of GPCRs in tumor development confound effective treatment; therefore, targeting a single common signaling component downstream of these receptors may be efficacious. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Hyperactive Gα_s signaling can mediate tumor progression in some tissues; however, recent work in medulloblastoma and basal cell carcinoma revealed that Gα_s can also function as a tumor suppressor in neoplasms derived from ectoderm cells including neural and epidermal stem/progenitor cells. In these stem-cell compartments, signaling through Gα_s suppresses self-renewal by inhibiting the Sonic Hedgehog (SHH) and Hippo pathways. The loss of GNAS, which encodes Gα_s, leads to activation of these pathways, over-proliferation of progenitor cells, and tumor formation. Gα_s activates the cAMP-dependent protein kinase A (PKA) signaling pathway and inhibits activation of SHH effectors Smoothened-Gli. In addition, Gα_s-cAMP-PKA activation negatively regulates the Hippo pathway by blocking the NF2-LATS1/2-Yap signaling. In this review, we will address the novel function of the signaling network regulated by Gα_s in suppression of SHH-driven tumorigenesis and the therapeutic approaches that can be envisioned to harness this pathway to inhibit tumor growth and progression.     

The Effects of Ribavirin on the GTP Level and the VIP Receptor Dynamic of Human IGR39 Cells

The discovery that arrestins can function as ligand-regulated signaling scaffolds has revealed a previously unappreciated level of complexity in G protein-coupled receptor (GPCR) signal transduction. Because arrestin-bound GPCRs are uncoupled from G proteins, arrestin binding can be viewed as switching receptors between two temporally and spatially distinct signaling modes. Recent work has established two factors that underscore this duality of GPCR signaling and suggest it may ultimately have therapeutic significance. The first is that signaling by receptor-arrestin “signalsomes” does not require heterotrimeric G protein activation. The second is that arrestin-dependent signals can be initiated by pathway-specific “biased agonists,” creating the potential for drugs that selectively modulate different aspects of GPCR function. Currently, however, little is known about the physiological relevance of G protein-independent signals at the cellular or whole animal levels, and additional work is needed to determine whether arrestin pathway-selective drugs will find clinical application.     

G蛋白偶联受体与G蛋白相互作用的最新研究进展

传统观念认为,在激动剂作用下,G蛋白偶联受体(GPCRs)能够激活G蛋白的α亚基,从而使Gα亚基与Gβγ亚基分离,被激活的Gα亚基通过信号转导进一步参与细胞的生理过程。但是,最新研究发现GPCRs和G蛋白存在多种偶联关系,GPCRs不仅能够激活Gα亚基,还可以与Gβγ亚基相互靠近,甚至会使G蛋白亚基构象发生重排而不分离,这对于疾病发病机制的研究及新的药物靶点的发现具有重要意义。就GPCRs与G蛋白之间的相互作用以及最新研究技术作一简要综述。     

A concept for G protein activation by G protein-coupled receptor dimers: the transducin/rhodopsin interface.

G protein-coupled receptors (GPCRs) are ubiquitous and essential in modulating virtually all physiological processes. These receptors share a similar structural design consisting of the seven-transmembrane alpha-helical segments. The active conformations of the receptors are stabilized by an agonist and couple to structurally highly conserved heterotrimeric G proteins. One of the most important unanswered questions is how GPCRs couple to their cognate G proteins. Phototransduction represents an excellent model system for understanding G protein signaling, owing to the high expression of rhodopsin in rod photoreceptors and the multidisciplinary experimental approaches used to study this GPCR. Here, we describe how a G protein (transducin) docks on to an oligomeric GPCR (rhodopsin), revealing structural details of this critical interface in the signal transduction process. This conceptual model takes into account recent structural information on the receptor and G protein, as well as oligomeric states of GPCRs.     

Heptahelical terpsichory. Who calls the tune?

The discovery that arrestins can function as ligand-regulated signaling scaffolds has revealed a previously unappreciated level of complexity in G protein-coupled receptor (GPCR) signal transduction. Because arrestin-bound GPCRs are uncoupled from G proteins, arrestin binding can be viewed as switching receptors between two temporally and spatially distinct signaling modes. Recent work has established two factors that underscore this duality of GPCR signaling and suggest it may ultimately have therapeutic significance. The first is that signaling by receptor-arrestin "signalsomes" does not require heterotrimeric G protein activation. The second is that arrestin-dependent signals can be initiated by pathway-specific "biased agonists," creating the potential for drugs that selectively modulate different aspects of GPCR function. Currently, however, little is known about the physiological relevance of G protein-independent signals at the cellular or whole animal levels, and additional work is needed to determine whether arrestin pathway-selective drugs will find clinical application.     

A constitutively active Gα subunit provides insights into the mechanism of G protein activation

The activation of Gα subunits of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) is a critical event underlying a variety of biological responses. Understanding how G proteins are activated will require structural and biochemical analyses of GPCRs complexed to their G protein partners, together with structure-function studies of Gα mutants that shed light on the different steps in the activation pathway. Previously, we reported that the substitution of a glycine for a proline at position 56 within the linker region connecting the helical and GTP-binding domains of a Gα chimera, designated αT*, yields a more readily exchangeable state for guanine nucleotides. Here we show that GDP-GTP exchange on αT*(G56P), in the presence of the light-activated GPCR, rhodopsin (R*), is less sensitive to the β1γ1 subunit complex than to wild-type αT*. We determined the X-ray crystal structure for the αT*(G56P) mutant and found that the G56P substitution leads to concerted changes that are transmitted to the conformationally sensitive switch regions, the α4-β6 loop, and the β6 strand. The α4-β6 loop has been proposed to be a GPCR contact site that signals to the TCAT motif and weakens the binding of the guanine ring of GDP, whereas the switch regions are the contact sites for the β1γ1 complex. Collectively, these biochemical and structural data lead us to suggest that αT*(G56P) may be adopting a conformation that is normally induced within Gα subunits by the combined actions of a GPCR and a Gβγ subunit complex during the G protein activation event.     

A biased ligand for OXE-R uncouples Gα and Gβγ signaling within a heterotrimer

Differential targeting of heterotrimeric G protein versus β-arrestin signaling are emerging concepts in G protein-coupled receptor (GPCR) research and drug discovery, and biased engagement by GPCR ligands of either β-arrestin or G protein pathways has been disclosed. Herein we report on a new mechanism of ligand bias to titrate the signaling specificity of a cell-surface GPCR. Using a combination of biomolecular and virtual screening, we identified the small-molecule modulator Gue1654, which inhibits Gβγ but not Gα signaling triggered upon activation of Gα(i)-βγ by the chemoattractant receptor OXE-R in both recombinant and human primary cells. Gue1654 does not interfere nonspecifically with signaling directly at or downstream of Gβγ. This hitherto unappreciated mechanism of ligand bias at a GPCR highlights both a new paradigm for functional selectivity and a potentially new strategy to develop pathway-specific therapeutics.     

Subtype-dependent regulation of Gβγ signalling

G protein-coupled receptors (GPCRs) transmit information to the cell interior by transducing external signals to heterotrimeric G protein subunits, Gα and Gβγ subunits, localized on the inner leaflet of the plasma membrane. Though the initial focus was mainly on Gα-mediated events, Gβγ subunits were later identified as major contributors to GPCR-G protein signalling. A broad functional array of Gβγ signalling has recently been attributed to Gβ and Gγ subtype diversity, comprising 5 Gβ and 12 Gγ subtypes, respectively. In addition to displaying selectivity towards each other to form the Gβγ dimer, numerous studies have identified preferences of distinct Gβγ combinations for specific GPCRs, Gα subtypes and effector molecules. Importantly, Gβ and Gγ subtype-dependent regulation of downstream effectors, representing a diverse range of signalling pathways and physiological functions have been found. Here, we review the literature on the repercussions of Gβ and Gγ subtype diversity on direct and indirect regulation of GPCR/G protein signalling events and their physiological outcomes. Our discussion additionally provides perspective in understanding the intricacies underlying molecular regulation of subtype-specific roles of Gβγ signalling and associated diseases.