Use of cellular impedance to characterize ligand functional selectivity at G protein-coupled receptors

Les récepteurs couplés aux protéines G (RCPGs) représentent la plus grande famille de cibles thérapeutiques pour le traitement d’une panoplie de pathologies humaines. Bien que plusieurs décennies de recherche aient permis de façonner nos connaissances sur ces protéines membranaires, notre compréhension des déterminants moléculaires de leur activité signalétique reste encore limitée. De ces domaines de recherche, une avancée récente a mis à jour un nouveau phénomène, appelé sélectivité fonctionnelle des ligands, qui a bouleversé les paradigmes décrivant leu fonctionnement de ces récepteurs. Ce concept émane d’observations montrant que l’activité pharmacologique de certains ligands n’est pas nécessairement conservée sur tout le répertoire signalétiques connu du récepteur et peu se restreindre à l'activation sélective d’un sous-groupe de voies de signalisation.Ce nouveau modèle pharmacologique de l'activation des RCPG ouvre de nouvelles possibilités pour la découverte de médicaments plus efficace et sûr, ciblant les RCPGs. En effet, il permet la conception de molécules modulant spécifiquement les voies signalétiques d’intérêt thérapeutique, sans engager les autres voies qui pourraient mener à des effets secondaires indésirables ou de la tolérance. Cette thèse décrit l'utilisation d'une nouvelle approche sans marquage, basée sur la mesure du changement l'impédance cellulaire. Par la mesure des changements cellulaires, comme la morphologie, l’adhésion et/ou la redistribution des macromolécules, cette approche permet de mesurer de façon simultanée l'activité de plusieurs voies de signalisation impliqués dans ces réponses. Utilisant le récepteur β2-adrénergique (β2AR) comme modèle, nous avons démontré que les variations dans l’impédance cellulaire étaient directement liées à l’activation de multiples voies de signalisation suite à la stimulation du récepteur par son ligand. L’agoniste type du β2AR, l’isoprotérénol, s’est avéré induire une réponse d’impédance dose-dépendante constituée, dans le temps, de plusieurs caractéristiques distinctes pouvant être bloquées de façon compétitive par l’antagoniste ICI118,551 Par l’utilisation d’inhibiteurs sélectifs, nous avons été en mesure de déterminer la contribution de plusieurs voies signalétiques canoniques, comme les voies dépendantes de Gs et Gi, la production d’AMPc et l’activation de ERK1/2, sur ces changements. De plus, la dissection de la réponse d’impédance a permis d’identifier une nouvelle voie de mobilisation du Ca2+ contribuant à la réponse globale des changements initiés par la stimulation du β2AR. Dans une autre étude, nous avons rapporté que la réponse calcique induite par le β2AR serait attribuable à une transactivation Gs-dépendant du récepteur purinergique P2Y11, lui-même couplé à la protéine Gq. La mesure d’impédance permettant de distinguer et de décrire une pléiade d’activités signalétiques, nous avons émis l’hypothèse que des ligands arborant des profils signalétiques différents généreraient des réponses d’impédance distinctes. Le criblage d’une librairie de ligands spécifiques au β2AR a révélé une grande variété de signatures d’impédance. Grâce au développement d’une approche computationnelle innovatrice, nous avons été en mesure de regrouper ces signatures en cinq classes de composés, un regroupement qui s’est avéré hautement corrélé avec le profil signalétique des différents ligands. Nous avons ensuite combiné le criblage de composés par impédance avec l’utilisation d’inhibiteurs sélectifs de voies signalétiques afin d’augmenter la résolution du regroupement. En évaluant l’impact d’une voie signalétique donnée sur la signature d’impédance, nous avons été en mesure de révéler une plus grande variété de textures parmi les ligands. De plus, cette méthode s’est avérée efficace pour prédire le profil signalétique d’une librairie de composés non caractérisés, ciblant le β2AR. Ces travaux ont mené à l’élaboration d’une méthode permettant d’exprimer visuellement la sélectivité fonctionnelle de ligands et ont révélé de nouvelles classes de composés pour ce récepteur. Ces nouvelles classes de composés ont ensuite été testées sur des cardiomyocytes humains, confirmant que les composés regroupés dans différentes classes produisent des effets distincts sur la contractilité de ces cellules. Globalement, ces travaux démontrent la pertinence de l’utilisation de l’impédance cellulaire pour une évaluation précise des différences fonctionnelles parmi les composés ciblant les RCPGs. En fournissant une représentation pluridimensionnelle de la signalisation émanant des RCPGs à l’aide d’un seul essai ne requérant pas de marquage, les signatures d’impédance représentent une stratégie simple et innovante pour l’évaluation de la fonctionnalité sélective des ligands. Cette méthode pourrait être d’une grande utilité dans le processus de découverte de nouveaux médicaments.

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding (K-obs) provide quite accurate measures of the agonist microscopic dissociation constant (K-A). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Efficacy can be quantitated in several ways based on functional data ( maximal effect of the agonist (E-max), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay ( K-obs/ EC50), a combined parameter EmaxKobs/EC50). Here I show that EmaxKobs/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

Trafficking and signaling of G protein-coupled receptors in the nervous system: implications for disease and therapy

G protein-coupled receptors (GPCRs) are expressed throughout the nervous system where they regulate multiple physiological processes, participate in neurological diseases, and are major targets for therapy. Given that many GPCRs respond to neurotransmitters and hormones that are present in the extracellular fluid and which do not readily cross the plasma membrane, receptor trafficking to and from the plasma membrane is a critically important determinant of cellular responsiveness. Moreover, trafficking of GPCRs throughout the endosomal system can initiate signaling events that are mechanistically and functionally distinct from those operating at the plasma membrane. This review discusses recent advances in the relationship between signaling and trafficking of GPCRs in the nervous system. It summarizes how receptor modifications influence trafficking, discusses mechanisms that regulate GPCR trafficking to and from the plasma membrane, reviews the relationship between trafficking and signaling, and considers the implications of GPCR trafficking to drug development.

Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors.

The extracellularly-responsive kinase (ERK) subfamily of mitogen-activated protein kinases (MAPKs) has been implicated in the regulation of cell growth and differentiation. Activation of ERKs involves a two-step protein kinase cascade lying upstream from ERK, in which the Raf family are the MAPK kinase kinases and the MEK1/MEK2 isoforms are the MAPK kinases. The linear sequence of Raf --> MEK --> ERK constitutes the ERK cascade. Although the ERK cascade is activated through growth factor-regulated receptor protein tyrosine kinases, they are also modulated through G protein-coupled receptors (GPCRs). All four G protein subfamilies (Gq/11 Gi/o, Gs and G12/13) influence the activation state of ERKs. In this review, we describe the ERK cascade and characteristics of its activation through GPCRs. We also discuss the identity of the intervening steps that may couple agonist binding at GPCRs to activation of the ERK cascade.

Irreversible and bivalent ligands for selected G-protein coupled receptors

Derivatives of pharmaceutical compounds that could potentially bind irreversibly to a hypertension controlling receptor were synthesised to provide tools for studying this receptor. Also compounds that simultaneously activate two cardiovascular receptors with opposing cellular mechanisms were synthesised. This resulted in a desired partial reduction in the activity of one receptor.

Functional characterisation of in vitro synthesised G-protein coupled receptors in polymersomes

Membrane proteins play an indispensable role in physiological processes. It is, therefore, not surprising that many diseases are based on the malfunction of membrane proteins. Hence membrane proteins and especially G-protein coupled receptors(GPCRs)- the largest subfamily- have become an important drug target. Due to their high selectivity and sensitivity membrane proteins are also feasible for the detection of small quantities of substances with biosensors. Despite this widespread interest in GPCRs due to their importance as drug targets and biosensors there is still a lack of knowledge of structure, function and endogenous ligands for quiet a few of the previously identified receptors.rnBottlenecks in over-expression, purification, reconstitution and handling of membrane proteins arise due to their hydrophobic nature. Therefore the production of reasonable amounts of functional membrane proteins for structural and functional studies is still challenging. Also the limited stability of lipid based membrane systems hampers their application as platforms forrnscreening applications and biosensors.rnIn recent years the in vitro protein synthesis became a promising alternative to gain better yields for expression of membrane proteins in bio-mimetic membrane systems. These expression systems are based on cell extracts. Therefore cellular effects on protein expression are reduced. The open nature of the cell-free expression systems easily allows for the adjustment of reactionrnconditions for the protein of interest. The cell-free expression in the presence of bio-mimetic membrane systems allows the direct incorporation of the membrane proteins and therefore skips the time-consuming purification and reconstitution processes. Amphiphilic block-copolymers emerged as promising alternative for the less stable lipid-based membrane systems. They, likernlipids, form membraneous structures in aqueous solutions but exhibit increased mechanical and chemical stability.rnThe aim of this work was the generation of a GPCR-functionalised membrane system by combining both promising alternatives: in vitro synthesis and polymeric membrane systems. This novel platform should be feasible for the characterisation of the incorporated GPCR. Immunodetection of Dopamine receptor 1 and 2 expressed in diblock- and triblock-polymersomes demonstrated the successful in vitro expression of GPCRs in polymeric membranes. Antibodyrnbinding studies suggested a favoured orientation of dopamine receptors in triblockpolymersomes.rnA dopamine-replacement assay on DRD2-functionalised immobilised triblockpolymersomes confirmed functionality of the receptor in the polymersomes. The altered binding curve suggests an effect of the altered hydrophobic environment presented by the polymer membrane on protein activity.

Induction of RAGE shedding by activation of G-protein-coupled receptors

The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimers disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cβ, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cβ, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca2+ signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNAmediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of soluble RAGE in the mouse lung. Our findings suggest that pharmacological stimulation of RAGE shedding might open alternative treatment strategies for Alzheimers disease and diabetes-induced inflammation.

Alternative splicing of pre-mRNA in cancer: focus on G protein-coupled peptide hormone receptors

Through alternative splicing, multiple different transcripts can be generated from a single gene. Alternative splicing represents an important molecular mechanism of gene regulation in physiological processes such as developmental programming as well as in disease. In cancer, splicing is significantly altered. Tumors express a different collection of alternative spliceoforms than normal tissues. Many tumor-associated splice variants arise from genes with an established role in carcinogenesis or tumor progression, and their functions can be oncogenic. This raises the possibility that products of alternative splicing play a pathogenic role in cancer. Moreover, cancer-associated spliceoforms represent potential diagnostic biomarkers and therapeutic targets. G protein-coupled peptide hormone receptors provide a good illustration of alternative splicing in cancer. The wild-type forms of these receptors have long been known to be expressed in cancer and to modulate tumor cell functions. They are also recognized as attractive clinical targets. Recently, splice variants of these receptors have been increasingly identified in various types of cancer. In particular, alternative cholecystokinin type 2, secretin, and growth hormone-releasing hormone receptor spliceoforms are expressed in tumors. Peptide hormone receptor splice variants can fundamentally differ from their wild-type receptor counterparts in pharmacological and functional characteristics, in their distribution in normal and malignant tissues, and in their potential use for clinical applications.

Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43

Butyrate is a short-chain fatty acid (SCFA) closely related to the ketone body ß-hydroxybutyrate (BHB), which is considered to be the major energy substrate during prolonged exercise or starvation. During fasting, serum growth hormone (GH) rises concomitantly with the accumulation of BHB and butyrate. Interactions between GH, ketone bodies and SCFA during the metabolic adaptation to fasting have been poorly investigated to date. In this study, we examined the effect of butyrate, an endogenous agonist for the two G-protein-coupled receptors (GPCR), GPR41 and 43, on non-stimulated and GH-releasing hormone (GHRH)-stimulated hGH secretion. Furthermore, we investigated the potential role of GPR41 and 43 on the generation of butyrate-induced intracellular Ca2+ signal and its ultimate impact on hGH secretion. To study this, wt-hGH was transfected into a rat pituitary tumour cell line stably expressing the human GHRH receptor. Treatment with butyrate promoted hGH synthesis and improved basal and GHRH-induced hGH-secretion. By acting through GPR41 and 43, butyrate enhanced intracellular free cytosolic Ca2+. Gene-specific silencing of these receptors led to a partial inhibition of the butyrate-induced intracellular Ca2+ rise resulting in a decrease of hGH secretion. This study suggests that butyrate is a metabolic intermediary, which contributes to the secretion and, therefore, to the metabolic actions of GH during fasting.

Specificity of somatostatin receptor and G -protein interactions as characterized by somatostatin receptor subtype specific antibodies

The neuropeptide somatostatin is a widely distributed general inhibitor of endocrine, exocrine, gastrointestinal and neural functions. The biological actions of somatostatin are initiated by interaction with high affinity, plasma membrane somatostatin receptors (sst receptors). Five sst receptor subtypes have been cloned and sequence analysis shows they are all members of the G protein coupled receptor superfamily. The G proteins play a pivotal role in sst receptor signal transduction and the specificity of somatostatin receptor-G protein coupling defines the possible range of cellular responses. However, the data for endogenous sst receptor and G protein coupling is very limited, and even when it is available, the sst receptor subtypes involved in G protein coupling and signal transduction are unknown due to the expression of multiple sst receptor subtypes in target cell lines or tissues of somatostatin.^ In an effort to characterize each individual sst receptor subtypes, antisera against unique C-terminal regions of different sst receptor subtypes have been developed in our lab. In this report, antisera made against the sst1, sst2A and sst4 receptors are characterized. They are highly specific to their corresponding receptors and efficiently immunoprecipitate the sst receptors. Using these antibodies, the cell lines expressing these sst receptor subtypes were identified with both immunoprecipitation and Western blot methods. The development of sst receptor subtype specific antibodies make it possible to determine the specificity of the sst receptor subtype and G protein coupling in target cells or tissues expressing multiple sst receptors, two questions were addressed by this thesis: (1) whether different cellular environments affect receptor subtype and G protein coupling; (2) whether different sst receptors couple to different G proteins in similar cellular environments.^ Taken together our findings, both sst1 and sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells, G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in GH$\sb4$C$\sb1$ cells. Further, sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in AR4-2J cells while sst4 receptors couple with G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells. Therefore, the G protein coupling of the same sst receptors in different cell lines is basically similar in that they all couple with multiple $\alpha$-subunits of the G$\rm \sb{i}$ proteins, suggesting cellular environment has little effect on receptor and G protein coupling. Moreover, different sst receptors have similar G protein coupling specificities in the same cell line, suggesting components other than receptor and G$\alpha$ subunits in the signal transduction pathways may contribute to specific functions of each sst receptor subtype. This series of experiments represent a novel approach in dissecting signal transduction pathways and may have general application in the field. Furthermore, this is the first systematic study of sst receptor subtype and G protein $\alpha$-subunit interaction in both transfected cells and in normal cell lines. The information generated will be very useful in our understanding of sst receptor signal transduction pathways and in directing future sst receptor research. ^

Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the α2A adrenergic receptor (α2A AR), with ERK activation mediated by the β2 adrenergic receptor (β2 AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the α2A AR, like that mediated by both the β2 AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant β-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both α2A and β2 AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the β2 AR, the α2A AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although β2 AR stimulation leads to redistribution of both the β2 AR and EGFR, activation of the α2A AR leads to redistribution of the EGFR but the α2A AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.

GIPC and GAIP Form a Complex with TrkA: A Putative Link between G Protein and Receptor Tyrosine Kinase Pathways

NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-γ1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways.

Identification of a receptor/G-protein contact site critical for signaling specificity and G-protein activation.

Each G protein-coupled receptor recognizes only a distinct subset of the many structurally closely related G proteins expressed within a cell. How this selectively is achieved at a molecular level is not well understood, particularly since no specific point-to-point contact sites between a receptor and its cognate G protein(s) have been identified. In this study, we demonstrate that a 4-aa epitope on the m2 muscarinic acetylcholine receptor, a prototypical Gi/o-coupled receptor, can specifically recognize the C-terminal 5 aa of alpha subunits of the Gi/o protein family. The m2 receptor residues involved in this interaction are predicted to be located on one side of an alpha-helical receptor region present at the junction between the third intracellular loop and the sixth transmembrane domain. Coexpression studies with hybrid m2/m3 muscarinic receptors and mutant G-protein alpha q subunits showed that the receptor/G-protein contact site identified in this study is essential for coupling specificity and G-protein activation.

RAMPs and the modulation of G-protein coupled receptors

Receptor activity modifying proteins (RAMPs) are a family of three proteins that can interact with G-protein coupled receptors (GPCRs) to create new receptors and also alter the signalling and trafficing of existing receptors. There are frequently changes in RAMP expression in cardiovascular disease RAMPs represent important drug targets.