Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators

The recent discovery of positive allosteric modulators (PAMs) for G-protein-coupled receptors open new possibilities to control a number of physiological and pathological processes. Understanding the mechanism of action of such compounds will provide new information on the activation process of these important receptors. Within the last 10 years, a number of studies indicate that G-protein-coupled receptors can form dimers, but the functional significance of this phenomenon remains elusive. Here we used the metabotropic glutamate receptors as a model, because these receptors, for which PAMs have been identified, are constitutive dimers. We used the quality control system of the GABA(B) receptor to generate metabotropic glutamate receptor dimers in which a single subunit binds a PAM. We show that one PAM/dimer is sufficient to enhance receptor activity. Such a potentiation can still be observed if the subunit unable to bind the PAM is also made unable to activate G-proteins. However, the PAM acts as a non-competitive antagonist when it binds in the subunit that cannot activate G-proteins. These data are consistent with a single heptahelical domain reaching the active state per dimer during receptor activation.     

C 族GPCRs 的半胱氨酸富集区的研究进展及药学意义

C族GPCRs是体内重要的受体,参与众多重要的生理和病理进程,并具有复杂的结构和激活机制。在体内该族受体形成组成性的二聚体并具有七螺旋跨膜结构(heptahelical transmembrane domain,HD)、捕蝇草模块(venus flytrap domain,VFT)和半胱氨酸富集区(cysteine-rich domain,CRD)。本文系统介绍了近年来CRD单体的序列和结构解析,以及参与受体激活过程的机制研究的历程和进展。同时也展望了这些基础研究成果对于开发新的更具有成药性的以C族GPCRs为靶点的变构剂的指导意义。     

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.     

G-protein-coupled receptor kinase specificity for beta-arrestin recruitment to the beta2-adrenergic receptor revealed by fluorescence resonance energy transfer

The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with beta-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and beta-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting beta-arrestins to specific receptors has not been elucidated. Here we use the beta(2)-adrenergic receptor (beta(2)AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the beta(2)AR and beta-arrestins. We show that beta(2)AR phosphorylation is required for high affinity beta-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote beta-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for beta-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that beta-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the beta(2)AR and induce beta-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures beta-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of beta-arrestin recruitment.     

C 族GPCRs 寡聚体的激活机制

C族GPCRs是体内重要的受体亚家族,参与众多重要的生理和病理进程。该族受体的单体包含七螺旋跨膜结构(heptahelical transmembrane domain,HD)、捕蝇草模块(venus flytrap domain,VFT)和半胱氨酸富集区(cysteine-rich domain,CRD)、C末端等多个功能域,同时形成组成性的二聚体和寡聚体。这种多功能域寡聚体结构使该家族受体的激活机制非常复杂,本文回顾和介绍了多年以来对该家族受体在单体、二聚体、寡聚体等多个层面的激活机制研究的历程和进展,同时也展望了进一步的研究方向和这些研究成果对于开发新型药物的意义。     

Identification of a preassembled TRH receptor-G(q/11) protein complex in HEK293 cells

Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gβ proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.     

Cell signalling through phospholipid breakdown

There is much evidence that G-proteins transduce the signal from receptors for Ca²⁺-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein -subunits. It is proposed that this protein is the a-subunit of the G-protein that regulates the phospholipase in this tissue.Ca²⁺-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca²⁺ protein kinase C and perhaps growth factor receptor tyrosine kinases.     

IP(3) receptor type 3 and PLCbeta2 are co-expressed with taste receptors T1R and T2R in rat taste bud cells

The Ca(2+) signaling cascade has been reported to be activated by many tastants in vertebrate taste systems. Recently we have shown that G(i2) and phospholipase Cbeta2 (PLCbeta2) are co-expressed in a subset of taste bud cells and are possibly involved in Ca(2+) triggering of taste signaling in rats. We report here that, as a component downstream of PLCbeta2, the type 3 isoform of the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R3) is specifically expressed in the same cells as PLCbeta2 in rat taste buds. We also show that cells expressing rT2R9, a probable cycloheximide receptor, are included among PLCbeta2- and IP(3)R3-positive cells, as in the case of rT1R2, a different type of taste receptor. Our findings indicate that PLCbeta2 and IP(3)R3 co-localize together with G(i2) as downstream components of two different types of taste receptors, T1R and T2R, in taste bud cells.     

The activation mechanism of class-C G-protein coupled receptors

Class-C G-protein coupled receptors (GPCRs) represent a distant group among the large family of GPCRs. This class includes the receptors for the main neurotransmitters, glutamate and gamma-aminobutyric acid (GABA), and the receptors for Ca(2+), some taste and pheromone molecules, as well as some orphan receptors. Like any other GPCRs, class-C receptors possess a heptahelical domain (HD) involved in heterotrimeric G-protein activation, but most of them also have a large extracellular domain (ECD) responsible for agonist recognition and binding. In addition, it is now well accepted that these receptors are dimers, either homo or heterodimers. This complex architecture raises a number of important questions. Here we will discuss our view of how agonist binding within the large ECD triggers the necessary change of conformation, or stabilize a specific conformation, of the heptahelical domain leading to G-protein activation. How ligands acting within the heptahelical domain can change the properties of these complex macromolecules.     

Dynamic integration of alpha-adrenergic and cholinergic signals in the atria: role of G protein-regulated inwardly rectifying K+ channels

Numerous heptahelical receptors use activation of heterotrimeric G proteins to convey a multitude of extracellular signals to appropriate effector molecules in the cell. Both high specificity and correct integration of these signals are required for reliable cell function. Yet the molecular machineries that allow each cell to merge information flowing across different receptors are not well understood. Here we demonstrate that G protein-regulated inwardly rectifying K(+) (GIRK) channels can operate as dynamic integrators of alpha-adrenergic and cholinergic signals in atrial myocytes. Acting at the last step of the cholinergic signaling cascade, these channels are activated by direct interactions with betagamma subunits of the inhibitory G proteins (G betagamma), and efficiently translate M(2) muscarinic acetylcholine receptor (M2R) activation into membrane hyperpolarization. The parallel activation of alpha-adrenergic receptors imposed a distinctive "signature" on the function of M2R-activated GIRK1/4 channels, affecting both the probability of G betagamma binding to the channel and its desensitization. This modulation of channel function was correlated with a parallel depletion of G beta and protein phosphatase 1 from the oligomeric GIRK1 complexes. Such plasticity of the immediate GIRK signaling environment suggests that multireceptor integration involves large protein networks undergoing dynamic changes upon receptor activation.     

The trick of the tail: protein-protein interactions of metabotropic glutamate receptors

It was initially believed that G-protein-coupled receptors, such as metabotropic glutamate receptors, could simply be described as individual proteins that are associated with intracellular signal cascades via G-proteins. This view is no longer tenable. Today we know that metabotropic glutamate receptors (mGluRs) can dimerize and bind to a variety of proteins in addition to trimeric G-proteins. These newly identified protein interactions led to the discovery of new regulatory mechanisms that are independent of and sometimes synergistic with the classical G-protein-coupled second messenger pathways. Notably, several of these mechanisms connect mGluR-mediated signaling to other receptor classes, thereby creating a network of different receptor types and associated signal cascades. The intracellular C-termini of mGluRs play a key role in the regulation of these networks, and various new protein interactions of these domains were described recently. Because mGluRs are involved in a variety of physiological and pathophysiological processes, some of the proteins interacting with this receptor class have potential as valuable pharmaceutical targets. This review will give a comprehensive overview of proteins interacting with mGluR C-termini, highlight new evolving regulatory mechanisms for glutamatergic signal transduction and discuss possibilities for future drug development.     

G-protein-coupled receptor agonists activate endogenous phospholipase Cepsilon and phospholipase Cbeta3 in a temporally distinct manner

Phospholipase Cepsilon (PLCepsilon) is one of the newest members of the phosphatidylinositol-specific phospholipase C (PLC) family. Previous studies have suggested that G-protein-coupled receptors (GPCRs) stimulate phosphoinositide (PI) hydrolysis by activating PLCbeta isoforms through G(q) family G proteins and Gbetagamma subunits. Using RNA interference to knock down PLC isoforms, we demonstrate that the GPCR agonists endothelin (ET-1), lysophosphatidic acid (LPA), and thrombin, acting through endogenous receptors, couple to both endogenous PLCepsilon and the PLCbeta isoform, PLCbeta3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist- and isoform-specific profiles. PLCbeta3 is activated acutely within the first minute of ET-1, LPA, or thrombin stimulation but does not contribute to sustained PI hydrolysis induced by LPA or thrombin and accounts for only part of ET-1 sustained stimulation. PLCepsilon, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLCepsilon in knockdown cells dose-dependently increases sustained, but not acute, agonist-stimulated PI hydrolysis. Furthermore, combined knockdown of both PLCepsilon and PLCbeta3 additively inhibits PI hydrolysis, suggesting independent regulation of each isoform. Importantly, ubiquitination of inositol 1,4,5-trisphosphate receptors correlates with sustained, but not acute, activation of PLCepsilon or PLCbeta3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLCepsilon and PLCbeta3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist-specific temporal profiles; however, PLCbeta3 is predominantly involved in acute and PLCepsilon in sustained PI hydrolysis.     

The human serotonin<Subscript>1A</Subscript> receptor exhibits G-protein-dependent cell surface dynamics

Seven transmembrane domain G-protein-coupled receptors constitute the largest family of proteins in mammals. Signal transduction events mediated by such receptors are the primary means by which cells communicate with and respond to their external environment. The major paradigm in this signal transduction process is that stimulation of the receptor leads to the recruitment and activation of heterotrimeric GTP-binding proteins. These initial events, which are fundamental to all types of G-protein-coupled receptor signaling, occur at the plasma membrane via protein–protein interactions. As a result, the dynamics of the activated receptor on cell surfaces represents an important determinant in its encounter with G-proteins, and has significant impact on the overall efficiency of the signal transduction process. We have monitored the cell surface dynamics of the serotonin_1A receptor, an important member of the G-protein-coupled receptor superfamily, in relation to its interaction with G-proteins. Fluorescence recovery after photobleaching experiments carried out with the receptor tagged to the enhanced yellow fluorescent protein indicate that G-protein activation alters the diffusion properties of the receptor in a manner suggesting the activation process leads to dissociation of G-proteins from the receptor. This result demonstrates that the cell surface dynamics of the serotonin_1A receptor is modulated in a G-protein-dependent manner. Importantly, this result could provide the basis for a sensitive and powerful approach to assess receptor/G-protein interaction in an intact cellular environment.     

Synergistic activation of phospholipases Cgamma and Cbeta: a novel mechanism for PI3K-independent enhancement of FcepsilonRI-induced mast cell mediator release

Antigen/IgE-mediated mast cell activation via FcvarepsilonRI can be markedly enhanced by the activation of other receptors expressed on mast cells and these receptors may thus contribute to the allergic response in vivo. One such receptor family is the G protein-coupled receptors (GPCRs). Although the signaling cascade linking FcvarepsilonRI aggregation to mast cell activation has been extensively investigated, the mechanisms by which GPCRs amplify this response are relatively unknown. To investigate this, we utilized prostaglandin (PG)E2 based on initial studies demonstrating its greater ability to augment antigen-mediated degranulation in mouse mast cells than other GPCR agonists examined. This enhancement, and the ability of PGE2 to amplify antigen-induced calcium mobilization, was independent of phosphoinositide 3-kinase but was linked to a pertussis toxin-sensitive synergistic translocation to the membrane of phospholipase (PL)Cgamma and PLCbeta and to an enhancement of PLCgamma phosphorylation. This "trans-synergistic" activation of PLCbeta and gamma, in turn, enhanced production of inositol 1,4,5-trisphosphate, store-operated calcium entry, and activation of protein kinase C (PKC) (alpha and beta). These responses were critical for the promotion of degranulation. This is the first report of synergistic activation between PLCgamma and PLCbeta that permits reinforcement of signals for degranulation in mast cells.     

Collision coupling, crosstalk, and compartmentalization in G-protein coupled receptor systems: can a single model explain disparate results?

The collision coupling model describes interactions between receptors and G-proteins as first requiring the molecules to find each other by diffusion. A variety of experimental data on G-protein activation have been interpreted as suggesting (or not) the compartmentalization of receptors and/or G-proteins in addition to a collision coupling mechanism. In this work, we use a mathematical model of G-protein activation via collision coupling but without compartmentalization to demonstrate that these disparate observations do not imply the existence of such compartments. In experiments with GTP analogs (commonly GTPγS), the extent of G-protein activation is predicted to be a function of both receptor number and the rate of GTP analog hydrolysis. The sensitivity of G-protein activation to receptor number is shown to be dependent upon the assay used, with the sensitivity of phosphate production assays (GTPase) >GTPγS-binding assays >cAMP inhibition assays. Finally, the amount of competition or crosstalk between receptor species activating the same type of G-proteins is predicted to depend on receptor and G-protein number, but in some (common) experimental regimes this dependence is expected to be minimal. Taken together, these observations suggest that the collision coupling model, without compartments of receptors and/or G-proteins, is sufficient to explain a variety of observations in literature data.     

Opioid receptor-coupled second messenger systems

Although pharmacological data provide strong evidence for different types of opioid receptors (e.g., mu, delta, and kappa), they share many common properties in their ability to couple to second messenger systems. All opioid receptor types are coupled to G-proteins, since agonist binding is diminished by guanine nucleotides and agonist-stimulated GTPase activity has been identified in several preparations. Moreover, all three types inhibit adenylyl cyclase. This second messenger system has been identified for opioid receptors in both isolated brain membranes and in transformed cell culture. Studies with chronic treatment with opioid agonists suggest that the coupling of receptors with G-proteins and second messenger effectors may play important roles in development of opioid tolerance.     

Calcium signaling and synaptic modulation: regulation of endocannabinoid-mediated synaptic modulation by calcium

Postsynaptic Ca2+ signal influences synaptic transmission through multiple mechanisms. Some of them involve retrograde messengers that are released from postsynaptic neurons in a Ca2+-dependent manner and modulate transmitter release through activation of presynaptic receptors. Recent studies have revealed essential roles of endocannabinoids in retrograde modulation of synaptic transmission. Endocannabinoid release is induced by either postsynaptic Ca2+ elevation alone or activation of postsynaptic Gq/11-coupled receptors with or without Ca2+ elevation. The former pathway is independent of phospholipase Cbeta (PLCbeta) and requires a large Ca2+ elevation to a micromolar range. The latter pathway requires PLCbeta and is facilitated by a moderate Ca2+ elevation to a submicromolar range. This facilitation is caused by Ca2+-dependency of receptor-driven PLCbeta activation. The released endocannabinoids then activate presynaptic cannabinoid receptor type 1 (CB1), and suppress transmitter release from presynaptic terminals. Both CB1 receptors and Gq/11-coupled receptors are widely distributed in the brain. Thus, the endocannabinoid-mediated retrograde modulation may be an important and widespread mechanism in the brain, by which postsynaptic events including Gq/11-coupled receptor activation and Ca2+ elevation can retrogradely influence presynaptic function.     

Oligomerization of opioid receptors

Opioid receptors belong to the family of G-protein-coupled receptors characterized by their seven transmembrane domains. The activation of these receptors by agonists such as morphine and endogenous opioid peptides leads to the activation of inhibitory G-proteins followed by a decrease in the levels of intracellular cAMP. Opioid receptor activation is also associated with the opening of K(+) channels and the inhibition of Ca(2+) channels. A number of investigations, prior to the development of opioid receptor cDNAs, suggested that opioid receptor types interacted with each other. Early pharmacological studies provided evidence for the probable interaction between opioid receptors. More recent studies using receptor selective antagonists, antisense oligonucleotides, or animals lacking opioid receptors further suggested that interactions between opioid receptor types could modulate their activity. We examined opioid receptor interactions using biochemical, biophysical, and pharmacological techniques. We used differential epitope tagging and selective immunoisolation of receptor complexes to demonstrate homotypic and heterotypic interactions between opioid receptor types. We also used the proximity-based bioluminescence resonance energy transfer assay to explore opioid receptor-receptor interactions in living cells. In this article we describe the biochemical and biophysical methods involved in the detection of receptor dimers. We also address some of the concerns and suggest precautions to be taken in studies examining receptor-receptor interactions.     

Gonadotropin-releasing hormone and the control of gonadotrope function

Normal gametogenesis and steroidogenesis is highly dependent on the pulsatile release of hypothalamic GnRH that binds high-affinity receptors present at the surface of pituitary gonadotrophs thereby triggering the synthesis and release of the gonadotropins LH and FSH. The mammalian GnRH receptor displays the classical heptahelical structure of G protein-coupled receptors with, however, a unique feature, the lack of a C-terminal tail. Accordingly, it does not desensitise sensu stricto, and internalises very poorly. It is now well established that GnRH stimulation induces the activation of a complex network of transduction pathways involved in the control of gonadotropin release and subunit gene expression. Other authors and ourselves have demonstrated that the GnRH action is associated with an increased complexity regarding gene regulation/cell function. Indeed GnRH affects the GnRH receptor gene itself and a number of additional genes that include some involved in cell signalling and auto-/paracrine regulation. The fact that GnRH regulates the expression of its own receptor, together with a host of other genes typically involved in its signal transduction cascades implies alteration/auto-adaptation in gonadotropic responsiveness. Furthermore, some of these genes respond differentially depending on whether the GnRH stimulation is intermittent or permanent suggesting specific roles in the dual process of activation/desensitisation. Thus, it can be assumed that the importance of pulsatility of GnRH action is closely related to, or dependent on, the inability of the GnRH receptor to desensitise. Moreover, multiple post-receptor events are crucial for both the regulation/plasticity of gonadotropic function and the maintenance of cell integrity.     

Calcium release at fertilization of Xenopus eggs requires type I IP(3) receptors, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.

Elevation of intracellular Ca2+ at fertilization is essential for the initiation of development in the Xenopus egg, but the pathway between sperm-egg interaction and Ca2+ release from the egg's endoplasmic reticulum is not well understood. Here we show that injection of an inhibitory antibody against the type I IP(3) receptor reduces Ca2+ release at fertilization, indicating that the Ca2+ release requires IP(3). We then examine how IP(3) production is initiated. Xenopus eggs were injected with specific inhibitors of the activation of two phospholipase C isoforms, PLCgamma and PLCbeta. The Src-homology 2 (SH2) domains of PLCgamma were used to inhibit SH2-mediated activation of PLCgamma, and an antibody against G(q) family G-proteins was used to inhibit G(q)-mediated activation of PLCbeta. Though the PLCgamma SH2 domains inhibited platelet-derived growth factor (PDGF)-induced Ca2+ release in eggs with exogenously expressed PDGF receptors, they did not inhibit the Ca2+ rise at fertilization. Similarly, the G(q) family antibody blocked serotonin-induced Ca2+ release in eggs with exogenously expressed serotonin 2C receptors, but not the Ca2+ rise at fertilization. A mixture of PLCgamma SH2 domains and the G(q) antibody also did not inhibit the Ca2+ rise at fertilization. These results indicate that Ca2+ release at fertilization of Xenopus eggs requires type I IP(3)-gated Ca2+ channels, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.