A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single beta2AR is incorporated per rHDL particle. The monomeric beta2AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.     

Biased signalling in follicle stimulating hormone action

Follicle-stimulating hormone (FSH) plays a crucial role in the control of reproduction by specifically binding to and activating a membrane receptor (FSHR) that belongs to the G protein-coupled receptor (GPCR) family. Similar to all GPCRs, FSHR activation mechanisms have generally been viewed as a two-state process connecting a unique FSH-bound active receptor to the Gs/cAMP pathway. Over the last decade, paralleling the breakthroughs that were made in the GPCR field, our understanding of FSH actions at the molecular level has dramatically changed. There are numerous facts indicating that the active FSHR is connected to a complex signalling network rather than the sole Gs/cAMP pathway. Consistently, the FSHR probably exists in equilibrium between multiple conformers, a subset of them being stabilized upon ligand binding. Importantly, the nature of the stabilized conformers of the receptor directly depends on the chemical structure of the ligand bound. This implies that it is possible to selectively control the intracellular signalling pathways activated by using biased ligands. Such biased ligands can be of different nature: small chemical molecules, glycosylation variants of the hormone or antibody/hormone complexes. Likewise, mutations or polymorphisms affecting the FSHR can also lead to stabilization of preferential conformers, hence to selective modulation of signalling pathways. These emerging notions offer a new conceptual framework that could potentially lead to the development of more specific drugs while also improving the way FSHR mutants/variants are functionally characterized.     

Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptor

The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, μ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.     

Beta-arrestin-mediated signaling in the heart

Beta-arrestin is a multifunctional adapter protein well known for its role in G-protein-coupled receptor (GPCR) desensitization. Exciting new evidence indicates that beta-arrestin is also a signaling molecule capable of initiating its own G-protein-independent signaling at GPCRs. One of the best-studied beta-arrestin signaling pathways is the one involving beta-arrestin-dependent activation of a mitogen-activated protein kinase cascade, the extracellular regulated kinase (ERK). ERK signaling, which is classically activated by agonist stimulation of the epidermal growth factor receptor (EGFR), can be activated by a number of GPCRs in a beta-arrestin-dependent manner. Recent work in animal models of heart failure suggests that beta-arrestin-dependent activation of EGFR/ERK signaling by the beta-1-adrenergic receptor, and possibly the angiotensin II Type 1A receptor, are cardioprotective. Hence, a new model of signaling at cardiac GPCRs has emerged and implicates classical G-protein-mediated signaling with promoting harmful remodeling in heart failure, while concurrently linking beta-arrestin-dependent, G-protein-independent signaling with cardioprotective effects. Based on this paradigm, a new class of drugs could be identified, termed "biased ligands", which simultaneously block harmful G-protein signaling, while also promoting cardioprotective beta-arrestin-dependent signaling, leading to a potential breakthrough in the treatment of chronic cardiac disease.     

Elucidation of signaling properties of vasopressin receptor-related receptor 1 by using the chimeric receptor approach

The identification of endogenous or surrogate ligands for orphan G protein-coupled receptors (GPCRs) represents one of the most important tasks in GPCR biology and pharmacology. The challenge lies in choosing an appropriate assay in the absence of ways to activate the receptor of interest. We investigated the signaling pathway for an orphan GPCR referred to here as vasopressin receptor-related receptor 1 (VRR1) by generating a chimeric receptor, V1a/VRR1. The engineered construct contained vasopressin V1a receptor with all three intracellular loops and C terminus replaced by those of VRR1. The chimera behaved like a typical GPCR when transiently and stably expressed in mammalian cell lines based on radioligand binding and receptor internalization studies. Upon arginine vasopressin stimulation, this chimeric receptor induced robust calcium mobilization and increase of adenylate cyclase activity. The observed signaling activities are through the activation of the chimera instead of endogenously expressed receptors, as single amino acid changes in the second transmembrane regions of the chimera drastically reduced receptor efficacy and potency. Our results suggest that VRR1 has dual signaling properties in coupling to both G(q) and G(S) pathways. Analysis of native VRR1 receptor signaling pathway by using a recently identified ligand for VRR1 confirmed this conclusion and therefore validated the utility of the chimeric receptor approach for signaling pathway identification.     

Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells

Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the alpha2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits alpha o, beta1, and gamma2 and a number of GPCRs including M4, alpha2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Galpha proteins shows specificity: there is interaction between the alpha2A receptor and Go, but little interaction between the alpha2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex.     

Engagement of β-arrestin by transactivated insulin-like growth factor receptor is needed for V2 vasopressin receptor-stimulated ERK1/2 activation

G protein-coupled receptors (GPCRs) have been shown to activate the mitogen-activated protein kinases, ERK1/2, through both G protein-dependent and -independent mechanisms. Here, we describe a G protein-independent mechanism that unravels an unanticipated role for β-arrestins. Stimulation of the V2 vasopressin receptor (V2R) in cultured cells or in vivo in rat kidney medullar collecting ducts led to the activation of ERK1/2 through the metalloproteinase-mediated shedding of a factor activating the insulin-like growth factor receptor (IGFR). This process was found to be both Src- and β-arrestin-dependent. Whereas Src was found to act upstream of the metalloproteinase activation and be required for the release of the IGFR-activating factor, β-arrestins were found to act downstream of the IGFR transactivation. Unexpectedly, the engagement of β-arrestins by the IGFR but not by the V2R was needed to promote the vasopressin-stimulated ERK1/2 activation, indicating that a pool of β-arrestins distinct from those β-arrestins recruited to the V2R acts downstream of the receptor tyrosine kinase to activate ERK1/2. Such a dual site of action for β-arrestins helps explain the pleiotropic actions of this scaffolding protein. Given the role that V2R-stimulated ERK1/2 plays in kidney cell proliferation, this transactivation mechanism may have important implications for renal pathophysiology. Still, the role of β-arrestins downstream of a transactivation event is not limited to the V2R, because we observed a similar involvement for an unrelated GPCR (the platelet-activating factor receptor), indicating that it may be a general mechanism shared among GPCRs.     

A functional transmembrane complex: The luteinizing hormone receptor with bound ligand and G protein

The luteinizing hormone receptor (LHR) is one of eight members in a cluster of the rhodopsin family of the large G protein-coupled receptor (GPCR) superfamily that contains some 800–900 genes in the human genome. LHR, along with its paralogons, follicle stimulating hormone receptor (FSHR) and thyroid stimulating hormone receptor, form one of the three classes in this cluster; the two other classes contain the relaxin-binding GPCRs and orphan GPCRs. These GPCRs are characterized by a relatively large ectodomain (ECD) containing leucine-rich-repeats (LRRs); in the class of glycoprotein hormone receptors, the LRR region is capped by N-terminal and C-terminal cysteine-rich regions. Binding of human chorionic gonadotropin (hCG) or luteinizing hormone to the LHR–ECD triggers a conformational change of the transmembrane region of the receptor facilitating binding and activation of Gs, followed by effector enzyme activation and subsequent intracellular signaling. Viewing LHR as a transmembrane anchoring protein that sequentially binds hCG and Gs to give the hCG–LHR–Gs complex, numerous interactions and conformational changes must be considered. There is, unfortunately, a paucity of structural data on LHR, but crystal structures exist for hCG, the homologous FSH–FSHR–ECD (N-terminal fragment) complex, rhodopsin (in the inactive state), an active form of Gαs (transducin), and the βγ heterodimer. Using a combined experimental (site-directed mutagenesis followed by characterization in transfected cells) and computational (homology modeling and molecular dynamics simulations) approach, good working models are being developed for the protein–protein interaction faces and, in some cases, the ensuing conformational changes induced by complex formation. hCG binding to the LHR–ECD appears to involve several LRRs; LHR activation can be described in terms of disrupting a network of H-bonds in the cytosolic halves of helices 1–3, 6, and 7; and binding of LHR to Gs involves, in large part, intracellular loop 2 binding, presumably to Gsα at its C-terminus. Major gaps exist in our understanding at the molecular level of the six-polypeptide chain complex, hCG–LHR–Gs, but considerable progress has been made in the past few years.     

β-Arrestin–biased signaling mediates memory reconsolidation

A long-standing hypothesis posits that a G protein-coupled signaling pathway mediates β-adrenergic nervous system functions, including learning and memory. Here we report that memory retrieval (reactivation) induces the activation of β₁-adrenergic β-arrestin signaling in the brain, which stimulates ERK signaling and protein synthesis, leading to postreactivation memory restabilization. β-Arrestin2-deficient mice exhibit impaired memory reconsolidation in object recognition, Morris water maze, and cocaine-conditioned place preference paradigms. Postreactivation blockade of both brain β-adrenergic Gs protein- and β-arrestin–dependent pathways disrupts memory reconsolidation. Unexpectedly, selective blockade of the Gs/cAMP/PKA signaling but not the β-arrestin/ERK signaling by the biased β-adrenergic ligands does not inhibit reconsolidation. Moreover, the expression of β-arrestin2 in the entorhinal cortex of β-arrestin 2–deficient mice rescues β₁-adrenergic ERK signaling and reconsolidation in a G protein pathway-independent manner. We demonstrate that β-arrestin–biased signaling regulates memory reconsolidation and reveal the potential for β-arrestin–biased ligands in the treatment of memory-related disorders.Alongside classical G protein pathways, activation of G protein-coupled receptors (GPCRs) stimulates β-arrestin–dependent signaling, leading to ERK phosphorylation and other downstream events (1, 2). Biased agonists, which induce functionally selective or biased receptor states and, thus, selectively activate one of the signaling pathways, have recently been identified for several GPCRs (3). Biased receptor agonism offers theoretical guidance for the discovery of a new generation of GPCR-targeted drugs with greater efficacy but fewer adverse effects. However, the lack of knowledge about the signaling pathways specifically eliciting a beneficial effect is a major obstacle in the understanding of disease mechanisms and the development of biased drugs targeting most GPCRs, especially those expressed in the central nervous system (CNS) with psychiatric importance.Besides their important roles in the cardiovascular and pulmonary systems, β-adrenergic receptors (β-ARs) are critically involved in CNS functions such as arousal, cognition, and stress-related behaviors (4, 5). β-Adrenergic neuronal signaling is important for neuroplasticity, including long-term potentiation (6) and memory formation (7). Accumulating cell biological evidence suggests that β-ARs also signal via G protein-independent, β-arrestin–dependent pathways (8–10). However, functions of β-AR in the CNS have been primarily ascribed to their classical role of stimulating Gs protein. The differential neurophysiological consequences for the G protein- and β-arrestin–dependent pathways, if any, have not been delineated.A longstanding hypothesis posits that a β-AR/Gs/protein kinase A (PKA) signaling pathway mediates memory reconsolidation (11–13), a process that strengthens, updates, or erases a previously acquired memory after recall (memory reactivation). This hypothesis is largely based on observations that β-ARs and molecules in the classical GPCR signaling pathway—such as cAMP (cAMP), PKA, and cAMP response element-binding protein (CREB)—are required for reconsolidation, which was determined by using receptor antagonists, kinase inhibitors, or gene knockout mice (11, 14, 15). Most of these molecules are also required for basal neural activity or plasticity, and there has been no direct evidence demonstrating that the function of β-ARs in reconsolidation is mediated by G protein/PKA or other signaling pathway (12). In the current study, we tested the potential involvement of G protein/cAMP/PKA-dependent pathway versus β-arrestin–dependent signaling in memory reconsolidation by using object recognition paradigm.     

Biased ligands for better cardiovascular drugs: dissecting G-protein-coupled receptor pharmacology

Drug discovery efforts targeting G-protein-coupled receptors (GPCR) have been immensely successful in creating new cardiovascular medicines. Currently marketed GPCR drugs are broadly classified as either agonists that activate receptors or antagonists that prevent receptor activation by endogenous stimuli. However, GPCR couple to a multitude of intracellular signaling pathways beyond classical G-protein signals, and these signals can be independently activated by biased ligands to vastly expand the potential for new drugs at these classic targets. By selectively engaging only a subset of a receptor's potential intracellular partners, biased ligands may deliver more precise therapeutic benefit with fewer side effects than current GPCR-targeted drugs. In this review, we discuss the history of biased ligand research, the current understanding of how biased ligands exert their unique pharmacology, and how research into GPCR signaling has uncovered previously unappreciated capabilities of receptor pharmacology. We focus on several receptors to illustrate the approaches taken and discoveries made, and how these are steadily illuminating the intricacies of GPCR pharmacology. Discoveries of biased ligands targeting the angiotensin II type 1 receptor and of separable pharmacology suggesting the potential value of biased ligands targeting the β-adrenergic receptors and nicotinic acid receptor GPR109a highlight the powerful clinical promise of this new category of potential therapeutics.     

Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors

Beta-arrestins critically regulate G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors (7TMRs), both by inhibiting classical G protein signaling and by initiating distinct beta-arrestin-mediated signaling. The recent discovery of beta-arrestin-biased ligands and receptor mutants has allowed characterization of these independent "G protein-mediated" and "beta-arrestin-mediated" signaling mechanisms of 7TMRs. However, the molecular mechanisms underlying the dual functions of beta-arrestins remain unclear. Here, using an intramolecular BRET (bioluminescence resonance energy transfer)-based biosensor of beta-arrestin 2 and a combination of biased ligands and/or biased mutants of three different 7TMRs, we provide evidence that beta-arrestin can adopt multiple "active" conformations. Surprisingly, phosphorylation-deficient mutants of the receptors are also capable of directing similar conformational changes in beta-arrestin as is the wild-type receptor. This indicates that distinct receptor conformations induced and/or stabilized by different ligands can promote distinct and functionally specific conformations in beta-arrestin even in the absence of receptor phosphorylation. Our data thus highlight another interesting aspect of 7TMR signaling--i.e., functionally specific receptor conformations can be translated to downstream effectors such as beta-arrestins, thereby governing their functional specificity.     

The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.     

Elucidation of G-protein and β-arrestin functional selectivity at the dopamine D2 receptor

The neuromodulator dopamine signals through the dopamine D2 receptor (D₂R) to modulate central nervous system functions through diverse signal transduction pathways. D₂R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D₂R signals through distinct G-protein and β-arrestin pathways, and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D₂R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D₂R mutants with strongly biased signal transduction for either the G-protein or β-arrestin interactions. These mutants were used to resolve the role of G proteins and β-arrestins in D₂R signaling assays. The results show that D₂R interactions with the two downstream effectors are dissociable and that G-protein signaling accounts for D₂R canonical MAP kinase signaling cascade activation, whereas β-arrestin only activates elements of this cascade under certain conditions. Nevertheless, when expressed in mice in GABAergic medium spiny neurons of the striatum, the β-arrestin–biased D₂R caused a significant potentiation of amphetamine-induced locomotion, whereas the G protein-biased D₂R had minimal effects. The mutant receptors generated here provide a molecular tool set that should enable a better definition of the individual roles of G-protein and β-arrestin signaling pathways in D₂R pharmacology, neurobiology, and associated pathologies.G protein-coupled receptors (GPCRs) are the largest receptor family and transmit the physiological effects of numerous biologically active molecules. GPCR signal transduction cascades account for diverse genomic, biochemical, cellular, and behavioral responses including cell fate determination, developmental reprogramming, olfactory, taste and light sensation, as well as complex behaviors mediated by neuromodulators (1). The diversity of responses to a particular hormone or neuromodulator is dictated not only by its cognate receptor but also by the ability of that receptor to engage distinct signaling pathways. For a number of GPCRs, their propensity to activate distinct G proteins can elicit diverse responses depending on the cellular environment (2). However, an even more subtle but intriguing mode of signaling has been attributed to the ability of a receptor to activate signaling pathways independent of G-protein activation, through the scaffolding of signaling complexes by β-arrestin, a component of the GPCR desensitization and internalization machinery (3). These two signaling modes harbor distinct functional properties, and in instances the same ligand can act as an agonist for one pathway but antagonist at the other. The selective or biased activation of a given pathway is commonly referred to as “functional selectivity” and can be easily demonstrated in heterologous systems especially when biased small molecule ligands are available (4). Biased GPCR ligands may have high therapeutic potential as these receptors represent the largest targets of drugs on the market. However, determining the functional contributions of G-protein and β-arrestin signaling pathways to the biological actions of an endogenous ligand acting upon its receptor still remains a challenging undertaking.Dopamine (DA) is a neuromodulator that is known to regulate movement, reward, cognition, emotion, and affect. The dopamine D2 receptor (D₂R) is a prominent GPCR that mediates the actions of DA. All typical antipsychotics, such as haloperidol, are potent D₂R blockers (5), whereas atypical antipsychotics, such as aripiprazole and clozapine, have unique pharmacology, exhibiting weak partial agonist activity at D₂R or reduced antagonist efficacy, respectively (6). Previous studies have demonstrated the ability of D₂Rs to engage different signal transduction pathways depending on the cellular complement of G proteins as well as their ability to regulate different physiological processes (7–9). β-arrestin 2 knockout mice provided robust behavioral and biochemical evidence for a critical D₂R/β-arrestin signaling pathway in the striatum (10). Furthermore, neuronal selective deletion of GSK3β, a putative D₂R/β-arrestin 2 effector, could reproduce the pharmacological blockade of D₂Rs with antipsychotics (11). Although these studies suggest that D₂Rs, like many other GPCRs, use pleiotropic signaling pathways to mediate their effects, the brain DA system is uniquely complex, as diverse responses may also rely upon many other determinants. One well-documented variable is the mode of stimulation of DA receptors, which is a function of the tonic or phasic release of DA (12). The expression profile of D₂R is also complex, being expressed not only in DA synthesizing neurons of the substantia nigra and ventral tegmental area where they function as presynaptic autoreceptors but also in GABAergic medium spiny neurons (MSNs), cholinergic interneurons of the striatum, and cortical neurons (13), where they function as postsynaptic receptors. Thus, understanding the contributions of functional selectivity at D₂R in intact biological systems is a challenge that cannot be elucidated in heterologous systems alone. To develop tools where this challenge can begin to be addressed, the Evolutionary Trace (ET) (14) approach was used to engineer D₂R mutants that selectively interact with either G proteins or β-arrestins, designated ^[Gprot]D₂R and ^[βarr]D₂R, respectively. These mutants show separation of G-protein and β-arrestin interactions, and expression of these mutants in vivo in the mouse striatum provides proof-of-concept for their biological activity and discrete functions.     

Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II

G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y(6.40) constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y223(5.58) in helix 5, Y306(7.53) of the NPxxY motif and R135(3.50) of the E(D)RY motif, highly conserved residues of the G protein binding site.     

G protein-coupled receptor oligomerization: implications for G protein activation and cell signaling

The cardiovascular system is richly endowed with G protein-coupled receptors (GPCRs), members of the largest family of plasma membrane-localized receptors. During the last 10 years, it has become increasingly clear that many, if not all, GPCRs function in oligomeric complexes, as either homo- or hetero-oligomers. This review explores the mechanistic implications of GPCR dimerization and/or oligomerization on receptor activation and interactions with G proteins. The effects of GPCR oligomerization on receptor pharmacology, GPCR-mediated signaling, and potential contributions to GPCR crosstalk will be considered in the context of receptors important in the cardiovascular system. Our evolving understanding of the structural and functional consequences of GPCR oligomerization may provide novel and more selective sites for pharmacological tuning of cardiovascular function.     

Activation mechanism of the β2-adrenergic receptor

A third of marketed drugs act by binding to a G-protein-coupled receptor (GPCR) and either triggering or preventing receptor activation. Although recent crystal structures have provided snapshots of both active and inactive functional states of GPCRs, these structures do not reveal the mechanism by which GPCRs transition between these states. Here we propose an activation mechanism for the β(2)-adrenergic receptor, a prototypical GPCR, based on atomic-level simulations in which an agonist-bound receptor transitions spontaneously from the active to the inactive crystallographically observed conformation. A loosely coupled allosteric network, comprising three regions that can each switch individually between multiple distinct conformations, links small perturbations at the extracellular drug-binding site to large conformational changes at the intracellular G-protein-binding site. Our simulations also exhibit an intermediate that may represent a receptor conformation to which a G protein binds during activation, and suggest that the first structural changes during receptor activation often take place on the intracellular side of the receptor, far from the drug-binding site. By capturing this fundamental signaling process in atomic detail, our results may provide a foundation for the design of drugs that control receptor signaling more precisely by stabilizing specific receptor conformations.     

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Galpha(s) protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive beta-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.     

Activation of G-protein-coupled receptors: a common molecular mechanism

G-protein-coupled receptors (GPCRs) are a large family of proteins that contain a seven transmembrane helical structural motif. They mediate responses to several ligands by binding and activating intracellular heterotrimeric G proteins. Since the cloning of the first GPCR, insights gained from structure–function studies, genetics and drug development have contributed to uncovering a common mechanism that explains the activation of diverse GPCRs by their cognate agonists. This mechanism takes into consideration the conservation of the structure–function relationship in the basic seven transmembrane structural motif, and the dynamic changes in receptor conformation that are associated with activation. Combining models derived from the X-ray structure of rhodopsin with structure–function data allows a deeper understanding of the activation mechanism of GPCRs.