G Protein-coupled Receptors Field Research Articles

Detecting and measuring of GPCR signaling - comparison of human induced pluripotent stem cells and immortal cell lines.

G protein-coupled receptors (GPCRs) are a large family of transmembrane proteins that play a major role in many physiological processes, and thus GPCR-targeted drug development has been widely promoted. Although research findings generated in immortal cell lines have contributed to the advancement of the GPCR field, the homogenous genetic backgrounds, and the overexpression of GPCRs in these cell lines make it difficult to correlate the results with clinical patients. Human induced pluripotent stem cells (hiPSCs) have the potential to overcome these limitations, because they contain patient specific genetic information and can differentiate into numerous cell types. To detect GPCRs in hiPSCs, highly selective labeling and sensitive imaging techniques are required. This review summarizes existing resonance energy transfer and protein complementation assay technologies, as well as existing and new labeling methods. The difficulties of extending existing detection methods to hiPSCs are discussed, as well as the potential of hiPSCs to expand GPCR research towards personalized medicine.

Deciphering Imidazoline Off-targets by Fishing in the Class A of GPCR field.

Based on the finding that a central antihypertensive agent with high affinity for I1-type imidazoline receptors - rilmenidine, shows cytotoxic effects on cultured cancer cell lines, it has been suggested that imidazoline receptors agonists might have a therapeutic potential in the cancer therapy. Nevertheless, potential rilmenidine side effects caused by activation of α-adrenoceptors, or other associated receptors and enzymes, might hinder its therapeutic benefits. Considering that human α-adrenoceptors belong to the rhodopsin-like class A of G-protein-coupled receptors (GPCRs) it is reasonable to assume that imidazolines might have the affinity for other receptors from the same class. Therefore, to investigate possible off-target effects of imidazoline ligands we have prepared a reverse docking protocol on class A GPCRs, using imidazoline ligands and their decoys. To verify our in silico results, three ligands with high scores and three ligands with low scores were tested for antagonistic activity on α2 - adrenoceptors.

Utilization of Biased G Protein-Coupled ReceptorSignaling towards Development of Safer andPersonalized Therapeutics.

G protein-coupled receptors (GPCRs) are involved in a wide variety of physiological processes. Therefore, approximately 40% of currently prescribed drugs have targeted this receptor family. Discovery of -arrestin mediated signaling and also separability of G protein and -arrestin signaling pathways have switched the research focus in the GPCR field towards development of biased ligands, which provide engagement of the receptor with a certain effector, thus enriching a specific signaling pathway. In this review, we summarize possible factors that impact signaling profiles of GPCRs such as oligomerization, drug treatment, disease conditions, genetic background, etc. along with relevant molecules that can be used to modulate signaling properties of GPCRs such as allosteric or bitopic ligands, ions, aptamers and pepducins. Moreover, we also discuss the importance of inclusion of pharmacogenomics and molecular dynamics simulations to achieve a holistic understanding of the relation between genetic background and structure and function of GPCRs and GPCR-related proteins. Consequently, specific downstream signaling pathways can be enriched while those that bring unwanted side effects can be prevented on a patient-specific basis. This will improve studies that centered on development of safer and personalized therapeutics, thus alleviating the burden on economy and public health.

Editorial: Monitoring endogenous GPCRs: lessons for drug design.

Editorial: Monitoring endogenous GPCRs: lessons for drug design.

On the Role of the Balance of GPCR Homo/ Heteroreceptor Complexes in the Brain

The early work on neuropeptide-monoamine receptor-receptor interactions in the Central Nervous System gave the first indications of the existence of G protein-coupled receptors (GPCRs) heteroreceptor complexes and the GPCR field began to expand from monomers into heteromers and higher order heteromers, including also GPCR-ion channel, Receptor Tyrosine Kinases (RTK)-GPCR and Receptor activity-modifying proteins-GPCR heteroreceptor complexes. The existence of heteroreceptor complexes with allosteric receptor-receptor interactions increases the diversity of receptor function including recognition, trafficking and signalling. We have proposed the molecular phenomenon of receptor-receptor interactions as a good way to understand of how brain function can increase through molecular integration of signals. An alteration in specific receptor-receptor interactions or their balance/equilibrium (with the corresponding monomers-homomers) are indeed considered to have a role in the pathogenic mechanisms that lead to various diseases, including drug addiction, depression, Parkinson's disease and schizophrenia. Therefore, targeting protomer-protomer interactions in heteroreceptor complexes or the balance with their corresponding homoreceptor complexes in discrete brain regions may become an important field for developing novel drugs, including heterobivalent drugs and optimal types of combined treatments. Increasing our understanding of molecular integration of signals via allosteric receptor-receptor interactions in the heteroreceptor complexes will have a major impact on the molecular medicine, leading to novel strategies for drug discovery and treatment of diseases.

Biased Signaling at Chemokine Receptors

The ability of G protein-coupled receptors (GPCRs) to activate selective signaling pathways according to the conformation stabilized by bound ligands (signaling bias) is a challenging concept in the GPCR field. Signaling bias has been documented for several GPCRs, including chemokine receptors. However, most of these studies examined the global signaling bias between G protein- and arrestin-dependent pathways, leaving unaddressed the potential bias between particular G protein subtypes. Here, we investigated the coupling selectivity of chemokine receptors CCR2, CCR5, and CCR7 in response to various ligands with G protein subtypes by using bioluminescence resonance energy transfer biosensors monitoring directly the activation of G proteins. We also compared data obtained with the G protein biosensors with those obtained with other functional readouts, such as β-arrestin-2 recruitment, cAMP accumulation, and calcium mobilization assays. We showed that the binding of chemokines to CCR2, CCR5, and CCR7 activated the three Gαi subtypes (Gαi1, Gαi2, and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies that generally correlate to their binding affinities. In addition, we showed that the binding of chemokines to CCR5 and CCR2 also activated Gα12, but not Gα13. For each receptor, we showed that the relative potency of various agonist chemokines was not identical in all assays, supporting the notion that signaling bias exists at chemokine receptors.

Les récepteurs couplés aux protéines G sous les feux de la rampe

G protein-coupled receptors (GPCRs), also known as seven transmembrane domain-spanning proteins (7TM), play an important role in tissue homeostasis and cellular and hormonal communication. GPCRs are targeted by a large panel of natural ligands such as photons, ions, metabolites, lipids and proteins but also by numerous drugs. Research efforts in the GPCR field have been rewarded in 2012 by the Nobel Price in Chemistry. The present article briefly summarizes our current knowledge on GPCRs and discusses future challenges in terms of fundamental aspects and therapeutic applications.

Molecular Dynamics Simulations of G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane-bound receptors with more than 800 members encoded by 351 genes in humans. It has been estimated that more than 50 % of clinically available drugs act on GPCRs, with an amount of 400, 50 and 25 druggable proteins for the class A, B and C, respectively. Furthermore, Class A GPCRs with approximately 25 % of marketed small drugs represent the most attractive pharmaceutical class. The recent availability of high-resolution 3-dimensional structures of some GPCRs supports the notion that GPCRs are dynamically versatile, and their functions can be modulated by several factors. In this scenario, molecular dynamics (MD) simulations techniques appear to be crucial when studying GPCR flexibility associated to functioning and ligand recognition. A general overview of biased and unbiased MD techniques is here presented with special emphasis on the recent results obtained in the GPCRs field.

Neuropeptide receptor positive allosteric modulation in epilepsy: Galanin modulation revealed

The therapeutic potential of neuropeptides has long been recognized; some successes have been registered, for example, in the tachykinin, endothelin, somatostatin, angiotensin, calcitonin gene-related peptide (CGRP), orexin, or glucagon-like peptide 1 (GLP1) fields (1), primarily in peripheral applications. However, given the nature of receptor–ligand interactions at the orthosteric site of G protein-coupled receptors (GPCRs), which seem to involve multiple pharmacophores that are hard to display on low-molecular-weight (LMW) ligands, it is not surprising that the number of such ligands, especially agonists, is very limited, particularly considering brain-penetrant ones. This is not because large pharmaceutical companies have neglected modern high throughput screening technology to find such drug candidates. Many synthetic neuropeptide analogs exist, but most of them are peptides, with inherent limitations in biovailability, plasma half-life/proteolytic degradation, brain penetration, and cost of goods. More recently, alternate approaches in the GPCR field emerged with negative or positive allosteric modulators (PAM), acting as antagonists or agonists, but only in the presence of the endogenous agonist. Galanin, which acts on three GPCRs (GalR1–3), has long been proposed to play a role in seizures; however, with few exceptions, the tools described are still peptides or peptidomimetics. Now, Lu et al. (2) report in PNAS on the discovery of CYM2503, an LMW, potent, selective, and in vivo active GalR2 PAM, effective in several modalities linked to epilepsy in rodents.

On the expanding terminology in the GPCR field: The meaning of receptor mosaics and receptor heteromers

The oligomerization of G protein-coupled receptors (GPCRs) is a fact that deserves further attention as increases both the complexity and diversity of the receptor-mediated signal transduction, thus enriching the cell signaling. Consequently, in the present review we tackle among others the problems concerning the terminology used to describe aspects surrounding the GPCRs oligomerization phenomenon. Therefore, the theoretical implications of the GPCR oligomerization will be briefly discussed together with possible implications of this phenomenon especially for new strategies in drug development.

Computational methods in drug design: Modeling G protein-coupled receptor monomers, dimers, and oligomers

G protein-coupled receptors (GPCRs) are membrane proteins that serve as very important links through which cellular signal transduction mechanisms are activated. Many vital physiological events such as sensory perception, immune defense, cell communication, chemotaxis, and neurotransmission are mediated by GPCRs. Not surprisingly, GPCRs are major targets for drug development today. Most modeling studies in the GPCR field have focused upon the creation of a model of a single GPCR (ie, a GPCR monomer) based upon the crystal structure of the Class A GPCR, rhodopsin. However, the emerging concept of GPCR dimerization has challenged our notions of the monomeric GPCR as functional unit. Recent work has shown not only that many GPCRs exist as homo- and heterodimers but also that GPCR oligomeric assembly may have important functional roles. This review focuses first on methodology for the creation of monomeric GPCR models. Special emphasis is given to the identification of localized regions where the structure of a GPCR may diverge from that of bovine rhodopsin. The review then focuses on GPCR dimers and oligomers and the bioinformatics methods available for identifying homo- and heterodimer interfaces.

Considerations in the design and use of chimeric G protein-coupled receptors

This chapter describes methods that can be employed to generate hybrid G protein-coupled receptors (GPCRs) and that are useful for the proper analysis of such mutant receptors. All examples discussed in this chapter involve the mutational analysis of different members of the muscarinic acetylcholine receptor family (M 1 –M 5 ). These receptors are ideally suited for hybrid receptor studies, as the five muscarinic receptor subtypes show distinct pharmacological and functional properties, while sharing a high degree of sequence homology. A meaningful interpretation of hybrid receptor data requires that experimental conditions are carefully chosen, such that differences in receptor numbers or improper receptor folding or localization do not interfere with proper data analysis. Given the extraordinary size of the GPCR superfamily and the ease with which chimeric GPCRs can be created by the use of modern PCR technology, hybrid GPCRs will continue to serve as important tools for researches in the GPCR field for many years to come.

STKE Focus Issue on GPCRs: The Evolution of G Protein-Coupled Receptor Signaling

G protein-coupled receptors (GPCRs) are one of the largest families of signaling proteins and are characterized by the presence of seven transmembrane domains and their ability to activate the heterotrimeric guanosine triphosphate (GTP) binding proteins (G proteins). Through the coupling to G proteins, GPCRs can mediate many different cellular effects and regulate processes as different as mitogenesis, cell movement, and cellular excitability. The original dogma about GPCR signaling was a fairly simple straightforward model in which each individual GPCR activated a single class of G protein. Furthermore, the G proteins had a high intrinsic GTPase activity and were initially believed not to utilize auxiliary regulators like those identified for the small GTPases. However, over time, the GPCR signaling field has gained in complexity, with GPCRs interacting with one another and other plasma membrane receptors, individual receptors coupling to more than one type of G protein, and the G proteins being regulated by GTPase activating proteins (GAPs). The STKE has information relevant to many aspects of G protein signaling and includes Reviews, Perspectives, Protocols, Connections Maps, relevant meetings in the Events section of the site, and other Web sites in the ST on the Web section of the site. If you are looking for others interested in G protein signaling, there are many people and labs in the Directory that list GPCRs as a focus of their research. Three new articles this issue highlight some of the changing views in the GPCR field. The ability and consequences of GPCRs coupling to more than one G protein is the subject of the review by Xiao ( ). GPCR multimerization is the topic of the perspective by Thelen and Baggiolini ([http://stke.sciencemag.org/cgi/content/full/sigtrans;2001/104/pe3][1]). The role of regulators of G protein signaling (RGS) in connecting GPCRs to other receptors and membrane proteins is the topic of the review by Druey ( ). Xiao focuses on the coupling of β-adrenergic receptors in the heart and the consequences of the dual coupling of the β2 subtype to both Gi and Gs pathways in terms of signal compartmentalization and cell survival signaling. These effects have important physiological ramifications when considering the underlying pathologies associated with cardiac heart failure and therapeutic approaches for treatment. GPCRs can oligomerize with other GPCR or with other types of receptors, such as ligand-gated ion channels. The direct interaction between GPCRs and ligand-gated ion channels is the topic of a perspective from the STKE archive by Brandon and Moss ( ). In this issue, Thelen and Bagglioni critically evaluate whether chemokine receptors heterodimerize or homodimerize and whether the multimerization is physiologically important. Druey focuses on the G proteins themselves. RGS proteins enhance the rate of GTPase activity of the Gα subunit. RGS proteins have additional protein-protein interaction motifs through which they couple G protein signaling to other membrane proteins, such as N-type calcium channels, receptor tyrosine kinases, and ephrin B proteins. For those interested in understanding the basics of GPCR signaling, the STKE has three Connections Maps by Pathway Authority R. Iyengar and members of his laboratory. These are Canonical Pathways that describe the signaling cascades initiated by Gs ([http://stke.sciencemag.org/cgi/cm/CMP_6634][2]), Gq ([http://stke.sciencemag.org/cgi/cm/CMP_6680][2]), and Gi ([http://stke.sciencemag.org/cgi/cm/CMP_7430][2]). The pathway highlighted in this issue, the Gαi Pathway ([http://stke.sciencemag.org/cgi/cm/CMP_7430][2]), describes the signaling from both the activated α subunits and the βγ subunits. Featured in This Focus Issue on GPCRs Related Resources at STKE [1]: http://stke.sciencemag.org/cgi/content/full/sigtrans;2001/104/pe4 [2]: pending:yes

Molecular Biology of Light Transduction by the Mammalian Photoreceptor, Rhodopsin

Rhodopsin, the vertebrate photoreceptor, is a prototypic molecule in the largest family of G- protein coupled receptors (GPCR). Like all receptors of this family, it contains three distinct domains: the cytoplasmic (intracellular) domain that is involved in all the protein-protein interactions; the transmembrane (TM) domain where the signal transduction begins, by light- catalysed isomerization of 11-cis-retinal to all trans-retinal, and the intradiscal domain which appears to be involved in a specific tertiary structure. The main focus of this talk is to describe efforts to understand specific structure and function in each domain. The main findings to be presented are as follows: 1. Intradiscal domain contains a globular tertiary structure. A central feature is a disulfide bond (Cys110-Cys187) which is conserved in most of the known GPCR. 2. The correct folding in vivo requires the formation of the above disulfide bond. Misfolding resulting in non-retinal binding is frequently caused by Retinitis Pigmentosa (RP) point mutations in the intradiscal and the TM domain. 3. In vivo folding studies, using RP mutations in every one of the seven helices, have shown that the packing of the helices in the TM domain and folding to form the intradiscal tertiary structure are coupled. 4. Cysteine mutagenesis has been used systematically to study the tertiary structure and light-dependent changes throughout the cytoplasmic face by combination of biochemical and biophysical studies. In particular, EPR spectroscopy following spin labeling of selected double cysteine mutants has shown movements in helices, including tilting, following retinal isomerization. 5. Large scale expression of mutants has allowed application of both 19F-NMR (solution) and MAS solid state NMR (in collaboration with Dr. Steve Smith's group, SUNY, Stony Brook). Results of current work are promising for detailed study of the conformational change. Finally, a unifying hypothesis, which is termed the central dogma in the GPCR field, will be proposed. This states that despite the enormous variation in “accessory” structural details, the principal mechanism of signal transduction starting with pertubation in the seven helical bundle is fundamentally the same in all GPCRs. Experiments to test helix movements, the first step in signal transduction following ligand binding in two adrenergic receptors are now feasible. The patterns of helix movements in them will be compared with the pattern demonstrated for rhodopsin and its mutants.