G-protein Coupled Receptor Superfamily Research Articles

Towards next generation adenosine A(2A) receptor antagonists.

The Adenosine A2A receptor is a member of the G-protein coupled receptor superfamily. It plays a key role in numerous physiological processes through the central nervous system and in peripheral tissues. Functional interactions between the A2A and dopamine D2 receptor has spurred interest in the use of antagonists as anti-Parkinson drugs. Additionally, oncology drugs are now being designed based on the potential for A2A antagonists to function as immunotherapeutics. From early studies based on classical xanthine type A2A antagonists through second generation agents, this mini review will cover aspects of the discovery, development, chemical synthesis and medicinal evaluation of this important class of drugs.

Melatonin in sperm biology: breaking paradigms.

Melatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT1 and MT2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORα1, RORα2 and RZRβ. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.

Oxytocin: Its Mechanism of Action and Receptor Signalling in the Myometrium

Oxytocin is a nonapeptide hormone that has a central role in the regulation of parturition and lactation. In this review, we address oxytocin receptor (OTR) signalling and its role in the myometrium during pregnancy and in labour. The OTR belongs to the rhodopsin-type (Class 1) of the G-protein coupled receptor superfamily and is regulated by changes in receptor expression, receptor desensitisation and local changes in oxytocin concentration. Receptor activation triggers a number of signalling events to stimulate contraction, primarily by elevating intracellular calcium (Ca(2+) ). This includes inositol-tris-phosphate-mediated store calcium release, store-operated Ca(2+) entry and voltage-operated Ca(2+) entry. We discuss each mechanism in turn and also discuss Ca(2+) -independent mechanisms such as Ca(2+) sensitisation. Because oxytocin induces contraction in the myometrium, both the activation and the inhibition of its receptor have long been targets in the management of dysfunctional and preterm labours, respectively. We discuss current and novel OTR agonists and antagonists and their use and potential benefit in obstetric practice. In this regard, we highlight three clinical scenarios: dysfunctional labour, postpartum haemorrhage and preterm birth.

Enantioselective synthesis of benzomorphan analogues by intramolecular oxa-Pictet–Spengler cyclization

A new strategy toward biologically active enantiomerically pure benzomorphan analogues is described. The key steps in the synthesis are the l-proline catalyzed asymmetric α-aminooxylation of an aldehyde and the titanium tetrachloride promoted intramolecular oxa-Pictet–Spengler cyclization of (4R)-2-(bromomethyl)-4-(2,5-dimethoxybenzyl)-1,3-dioxolane. In the intramolecular oxa-Pictet–Spengler cyclization, cis-pyran 8a (71%) formed over the trans-pyran 8b (14%). Computational modeling studies provided an insight into the stereoselectivity of the products. Docking studies of benzomorphan derivatives indicated that compound 6c had the best binding efficiency, and that it formed extensive hydrogen bonding with TYR148 on transmembrane (TM) helix 3 and HIS297 on TM6 of the μ-opioid receptor, which belong to the G-protein coupled receptor super family.

English

Melatonin (MLT) is a pleiotropic neurohormone controlling many physiological processes and whose dysfunction may contribute to several different diseases, such as neurodegenerative diseases, circadian and mood disorders, insomnia, type 2 diabetes and pain. Melatonin is synthesized by the pineal gland during the night and acts through 2 G-protein coupled receptors (GPCRs), MT1 (MEL1a) and MT2 (MEL1b). Although a bulk of research has examined the physiopathological effects of MLT, few studies have investigated the selective role played by MT1 and MT2 receptors. Here we have reviewed current knowledge about the implications of MT2 receptors in brain functions. We searched PubMed, Web of Science, Scopus, Google Scholar and articles' reference lists for studies on MT2 receptor ligands in sleep, anxiety, neuropsychiatric diseases and psychopharmacology, including genetic studies on the MTNR1B gene, which encodes the melatonin MT2 receptor. These studies demonstrate that MT2 receptors are involved in the pathophysiology and pharmacology of sleep disorders, anxiety, depression, Alzheimer disease and pain and that selective MT2 receptor agonists show hypnotic and anxiolytic properties. Studies examining the role of MT2 receptors in psychopharmacology are still limited. The development of novel selective MT2 receptor ligands, together with further preclinical in vivo studies, may clarify the role of this receptor in brain function and psychopharmacology. The superfamily of GPCRs has proven to be among the most successful drug targets and, consequently, MT2 receptors have great potential for pioneer drug discovery in the treatment of mental diseases for which limited therapeutic targets are currently available.

Chapter Ten - Constitutive Activity of Bitter Taste Receptors (T2Rs)

G protein-coupled receptors (GPCRs) play a vital role in transmitting an extracellular stimuli or signal into an intracellular response in various cells. In some scenarios, GPCRs or their mutants can also signal in the absence of an agonist or an external stimulus, referred to as basal or constitutive activity, and those mutants are termed constitutively active mutants (CAMs). Bitter taste is one of the five basic tastes and is mediated by bitter taste receptors (T2Rs), which belong to the GPCR superfamily. The 25 T2Rs present in humans do not belong to any of the major GPCR classes, and their classification is ambiguous. The characterization of T2Rs in many extraoral tissues including the airways and upper respiratory tract, where they were shown to cause bronchodilation and influence host susceptibility to infection, underscores the therapeutic relevance of these receptors. Recent structure-function and pharmacological studies on T2Rs led to the identification of CAMs. In this review, we summarize the major findings on constitutive activity of T2Rs and their diverse roles. We discuss the usefulness of the T2R CAMs in terms of the discovery of bitter taste blockers, elucidating the mechanisms of T2R activation and dissecting the physiological pathways.

The effect of the estrous cycle on the expression of prepro-orexin gene and protein and the levels of orexin A and B in the porcine pituitary

Hypothalamic peptides orexin A (OXA) and orexin B (OXB) are derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin (PPO). They act via two orexin receptors (OX1R and OX2R), which belong to the G-protein coupled receptor superfamily. Orexins are implicated in the regulation of arousal states, energy homeostasis and reproductive neuroendocrine function. The objective of this study was to investigate the presence and changes in orexin expression in the porcine pituitary during the estrous cycle. Adenohypophysis (AP) and neurohypophysis (NP) tissue samples were harvested on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the estrous cycle. The expression of the PPO gene increased in AP and NP during the estrous cycle. The highest PPO protein concentrations in AP were reported on days 2 to 3 (P<0.05), and in NP – on days 10 to 12 and 17 to 19 (P<0.05). The expression of PPO mRNA was lower in AP than in NP, but PPO protein levels were higher in AP. In AP, OXA immunoreactivity was higher (P<0.05) on days 10 to 12 and 14 to 16. In NP, the highest (P<0.05) content of the analyzed protein was observed on days 10 to 12 and the lowest (P<0.05) – on days 14 to 16 and 17 to 19. OXB immunoreactivity in AP reached the highest level (P<0.05) on days 2 to 3, and the lowest level (P<0.05) was determined on days 10 to 12 and 17 to 19. OXB protein concentrations in NP peaked (P<0.05) on days 10 to 12 of the cycle. Our study was the first experiment to demonstrate the expression of the orexin gene and orexin proteins in the porcine pituitary and the correlations between expression levels and the phase of the estrous cycle.

Interaction of methionine–enkephalins with raft-forming lipids: monolayers and BAM experiments

Enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are opioid peptides with proven antinociceptive action in organism. They interact with opioid receptors belonging to G-protein coupled receptor superfamily. It is known that these receptors are located preferably in membrane rafts composed mainly of sphingomyelin (Sm), cholesterol (Cho), and phosphatidylcholine. In the present work, using Langmuir's monolayer technique in combination with Wilhelmy's method for measuring the surface pressure, the interaction of synthetic methionine-enkephalin and its amidated derivative with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Sm, and Cho, as well as with their double and triple mixtures, was studied. From the pressure/area isotherms measured, the compressional moduli of the lipids and lipid-peptide monolayers were determined. Our results showed that the addition of the synthetic enkephalins to the monolayers studied led to change in the lipid monolayers characteristics, which was more evident in enkephalinamide case. In addition, using Brewster angle microscopy (BAM), the surface morphology of the lipid monolayers, before and after the injection of both enkephalins, was determined. The BAM images showed an increase in surface density of the mixed surface lipids/enkephalins films, especially with double and triple component lipid mixtures. This effect was more pronounced for the enkephalinamide as well. These observations showed that there was an interaction between the peptides and the raft-forming lipids, which was stronger for the amidated peptide, suggesting a difference in folding of both enkephalins. Our research demonstrates the potential of lipid monolayers for elegant and simple membrane models to study lipid-peptide interactions at the plane of biomembranes.

Serotonin in the pathophysiology and treatment of CNS disorders

Decades ago, in the 1950s, serotonin (5-hydroxytryptamine, 5-Ht) was discovered in the mammalian central and peripheral nervous systems, and successively, its role as an essential neurotransmitter was revealed. advances in understanding the effect of 5-Ht have represented one of the success stories of neuropharmacology. Since its discovery, an enormous amount of experimental evidence has revealed the pivotal role of this biogenic amine in a bewildering diversity of behavioural and physiological processes. this is not surprising, considering the large distribution of 5-Ht-containing axon terminals throughout the central nervous systems (CNS), although these monoamines are synthesized by a small group of neurons within the brain stem. the serotonergic system is one of the most diffusively organized projection systems of the mammalian brain. the majority of the neurons containing 5-Ht generically called the raphe nuclei are located in the brainstem and in some regions of the reticular formation. Recent receptor discoveries have permitted the identification and classification of up to seven families of 5-Ht receptors (5-Ht1–5-Ht7) to date. all 5-Ht receptors belong to the seven transmembrane domain G-proteincoupled receptor (GPCR) superfamily, except for the 5-Ht3 receptor which is a ligand-gated channel. the 5-Ht1 receptor class is comprised of five receptor subtypes (5-Ht1a, 5-Ht1B, 5-Ht1D, 5-Ht1E and 5-Ht1F), which, in humans, share 40–63 % overall sequence identity and couple preferentially, although not exclusively, to Gi/o to inhibit caMP formation. among the multiple classes of 5-Ht receptors described in the CNS, much attention has been devoted to the 5-Ht2 receptor family since it has been shown by experimental and clinical observation to represent a possible therapeutic this special issue is in honour of Ennio Esposito, who devoted his unfortunately short but productive scientific career to serotonin research and made significant contributions to the field of neuropharmacology of monoaminergic systems in neuropsychiatric disorders. after receiving his degree in Medicine at the University of Chieti, Ennio moved to Milan to the laboratory of Neuropharmacology at the Institute of Pharmacological Research Mario Negri, under the supervision of Rosario Samanin. Successively, Ennio did his postdoc in the USa. at the Department of Psychiatry and Pharmacology at Yale University School of Medicine, New Haven, in the laboratory of Dr. Benjamin S. Bunney. Back in Italy, in 1989, he joined the Consorzio Mario Negri Sud, a Biomedical and Pharmacological Research Centre at Santa Maria Imbaro, Chieti. In this new institution, a few miles from his hometown, he created and directed the laboratory of Neurophysiology for about 20 years. the aim of this special issue is to present recent developments in research areas Ennio has been particularly involved in, that is, the serotonin receptors, especially the 5-Ht2C subtype. It includes 18 papers written by several of his former students, co-workers and friends, as well as by distinguished international researchers with similar research interests.

Upregulation of GPR34 expression affects the progression and prognosis of human gastric adenocarcinoma by PI3K/PDK1/AKT pathway.

G-protein coupled receptor 34 (GPR34), which belongs to the G-protein coupled receptors superfamily, is reportedly expressed highly in the spread of several solid tumors. However, its expression in gastric primary tumor and potential role in gastric cancer development and progression have not been determined. Immunohistochemistry, real-time RT-PCR and western blot methods were used to determine GPR34 expression in human gastric cancer tissues/cell lines and matched adjacent tissues/ normal mucosal cell line. A statistical analysis was performed to establish the potential correlation between GPR34 expression and the patients' clinicopathological characteristics, tumor progression, and prognosis. Stably transfected NCI-N87 cell lines with either GPR34 over-expression or knock-down were constructed to determine the effect of GPR34 on gastric cancer cell invasion and migration, and to explain the preliminary molecular mechanism of GPR34 in gastric cancer metastasis. GPR34 is up-regulated in primary gastric cancer tissues/cell lines compared with matched adjacent tissues/normal mucosal cell line, and when the relationship between GPR34 expression and the the clinicopathological characteristics was analyzed, it was shown that GPR34 expression is significantly correlated with tumor differentiation, infiltration depth, and lymph node status and had a significant influence on prognosis. Furthermore, GPR34-overexpression increased while GPR34-knockdown inhibited NCI-N87 cell invasion in vitro by PI3K/PDK1/AKT pathway. Taken together, up-regulation of GPR34 expression in human gastric carcinoma may play a critical role in tumor progression and in determining patient prognosis. GPR34 may be a useful diagnostic or prognostic molecular biomarker, and a potential target for therapeutic intervention.

A synthetic bivalent ligand of CXCR4 inhibits HIV infection

G-protein-coupled receptors (GPCRs) are cell membrane protein receptors that transduce signals across the cell membrane and are important targets for therapeutic interventions. As members of the GPCR superfamily, chemokine receptors such as CXCR4 play critical roles in normal physiology as well as the pathology of many human diseases including cancer, inflammation, autoimmune diseases, and human immunodeficiency virus (HIV) infection. Here we report the discovery and study of a novel peptide ligand of CXCR4 using d-amino acids and bivalent ligand approach. This peptide, DV1-K-(DV3), shows very high affinity for CXCR4 with an IC50 of 4nM in anti-CXCR4 monoclonal antibody (mAb) 12G5 competitive assay, which is more potent than full length natural ligand SDF-1α, even though the peptide is less than half of the number of residues of SDF-1α. This peptide can block the calcium influx stimulated by SDF-1α and inhibit cancer cell migration in vitro via CXCR4, thus functioning as a CXCR4 antagonist. Furthermore, DV1-K-(DV3) peptide displayed anti-HIV activity by inhibiting HIV-1 infection mediated by CXCR4. With its high receptor affinity and stability from d-amino acids, this peptide may be a new probe of CXCR4 functions in physiology and pathology and promising lead for therapeutic development.

Characterization of the dynamic events of GPCRs by automated computational simulations

The recent advances in membrane protein crystallography have provided extremely valuable structural information of the superfamily of GPCRs (G-protein-coupled receptors). This has been particularly true for a few receptors whose structure was solved several times under different biochemical conditions. It follows that the mechanisms of receptor conformational equilibrium and related dynamic events can be explored by computational simulations. In the present article, we summarize our recent understanding of several dynamic features of GPCRs, accomplished through the use of MD (molecular dynamics) simulations. Our pipeline for the MD simulations of GPCRs, implemented in the web service http://gpcr.usc.es, is updated in the present paper and illustrated by recent applications. Special emphasis is put on the A2A adenosine receptor, one of the selected cases where crystal structures in several conformations and conditions exist, and on the dimerization process of the CXCR4 (CXC chemokine receptor 4).

Recent Advances in the Rationale Design of GPER Ligands

G-Protein Coupled Receptor (GPCR) superfamily, which comprises approximately 900 members, is the largest family of protein targets with proven therapeutic value. Although at least 500 GPCRs have been identified as therapeutically relevant, only thirteen GPCRs have been structurally characterized in apo-form or in complex with ligands. GPCRs share relatively low sequence similarity making hard the process of homology modelling, nevertheless some successful hits have been determined. Recently, the G-protein-coupled estrogen receptor 1 (GPER, formerly known as GPR30) has attracted increasing interest due to its ability in mediating estrogen signaling in different normal and cancer tissues. In this regard, the identification of selective GPER ligands has provided valuable tools in order to differentiate the specific functions elicited by this novel estrogen receptor respect to those exerted by the classical estrogen receptors (ERs). In this review, we focus on GPER examining "in silico" docking simulations and evaluating the different binding modes of diverse natural and synthetic ligands.

Recent Advances in the Rationale Design of GPER Ligands

G-Protein Coupled Receptor (GPCR) superfamily, which comprises approximately 900 members, is the largest family of protein targets with proven therapeutic value. Although at least 500 GPCRs have been identified as therapeutically relevant, only thirteen GPCRs have been structurally characterized in apo-form or in complex with ligands. GPCRs share relatively low sequence similarity making hard the process of homology modelling, nevertheless some successful hits have been determined. Recently, the G-proteincoupled estrogen receptor 1 (GPER, formerly known as GPR30) has attracted increasing interest due to its ability in mediating estrogen signaling in different normal and cancer tissues. In this regard, the identification of selective GPER ligands has provided valuable tools in order to differentiate the specific functions elicited by this novel estrogen receptor respect to those exerted by the classical estrogen receptors (ERs). In this review, we focus on GPER examining “in silico” docking simulations and evaluating the different binding modes of diverse natural and synthetic ligands.

Structure-Function of the G Protein–Coupled Receptor Superfamily

During the past few years, crystallography of G protein-coupled receptors (GPCRs) has experienced exponential growth, resulting in the determination of the structures of 16 distinct receptors-9 of them in 2012 alone. Including closely related subtype homology models, this coverage amounts to approximately 12% of the human GPCR superfamily. The adrenergic, rhodopsin, and adenosine receptor systems are also described by agonist-bound active-state structures, including a structure of the receptor-G protein complex for the β(2)-adrenergic receptor. Biochemical and biophysical techniques, such as nuclear magnetic resonance and hydrogen-deuterium exchange coupled with mass spectrometry, are providing complementary insights into ligand-dependent dynamic equilibrium between different functional states. Additional details revealed by high-resolution structures illustrate the receptors as allosteric machines that are controlled not only by ligands but also by ions, lipids, cholesterol, and water. This wealth of data is helping redefine our knowledge of how GPCRs recognize such a diverse array of ligands and how they transmit signals 30 angstroms across the cell membrane; it also is shedding light on a structural basis of GPCR allosteric modulation and biased signaling.

Hybrid Molecular Mechanics/Coarse-Grained Simulations for Structural Prediction of G-Protein Coupled Receptor/Ligand Complexes

Understanding how ligands bind to G-protein coupled receptors (GPCRs) provides insights into a myriad of cell processes and is crucial for drug development. Here we extend a hybrid molecular mechanics/coarse-grained (MM/CG) approach applied previously to enzymes to GPCR/ligand complexes. The accuracy of this method for structural predictions is established by comparison with recent atomistic molecular dynamics simulations on the human β2 adrenergic receptor, a member of the GPCRs superfamily. The results obtained with the MM/CG methodology show a good agreement with previous all-atom classical dynamics simulations, in particular in the structural description of the ligand binding site. This approach could be used for high-throughput predictions of ligand poses in a variety of GPCRs.

Structure prediction and molecular dynamics simulations of a G-protein coupled receptor: human CCR2 receptor

CC chemokine receptor type-2 (CCR2) is a member of G-protein coupled receptors superfamily, expressed on the cell surface of monocytes and macrophages. It binds to the monocyte chemoattractant protein-1, a CC chemokine, produced at the sites of inflammation and infection. A homology model of human CCR2 receptor based on the recently available C-X-C chemokine recepor-4 crystal structure has been reported. Ligand information was used as an essential element in the homology modeling process. Six known CCR2 antagonists were docked into the model using simple and induced fit docking procedure. Docked complexes were then subjected to visual inspection to check their suitability to explain the experimental data obtained from site directed mutagenesis and structure-activity relationship studies. The homology model was refined, validated, and assessed for its performance in docking-based virtual screening on a set of CCR2 antagonists and decoys. The docked complexes of CCR2 with the known antagonists, TAK779, a dual CCR2/CCR5 antagonist, and Teijin-comp1, a CCR2 specific antagonist were subjected to molecular dynamics (MD) simulations, which further validated the binding modes of these antagonists. B-factor analysis of 20 ns MD simulations demonstrated that Cys190 is helpful in providing structural rigidity to the extracellular loop (EL2). Residues important for CCR2 antagonism were recognized using free energy decomposition studies. The acidic residue Glu291 from TM7, a conserved residue in chemokine receptors, is favorable for the binding of Teijin-comp1 with CCR2 by ΔG of −11.4 kcal/mol. Its contribution arises more from the side chains than the backbone atoms. In addition, Tyr193 from EL2 contributes −0.9 kcal/mol towards the binding of the CCR2 specific antagonist with the receptor. Here, the homology modeling and subsequent molecular modeling studies proved successful in probing the structure of human CCR2 chemokine receptor for the structure-based virtual screening and predicting the binding modes of CCR2 antagonists.

Serotonergic signaling: multiple effectors and pleiotropic effects

AbstractSerotonin (5‐HT) is a neurotransmitter which exerts both central and peripheral control on numerous physiological functions such as sleep/wake cycle, thermoregulation, food intake, nociception, locomotion, sexual behavior, gastrointestinal motility, blood coagulation, and cardiovascular homeostasis. These multiple effects are mediated by a family of serotonin receptors which comprises at least 13 distinct G‐protein‐coupled receptors (GPCRs) of the A type and a combination of ligand‐gated cation channel heteropentameric receptors of the conserved 5‐HT3 Cys‐loop type. Many of these receptors are targets of pharmaceutical drugs justifying the wide interest to elucidate their functioning. Over the past decades, substantial investigations have led to the current understanding of the complex signal transduction mechanisms which these receptors initiate. The idea of a unique coupling of a receptor to a linear pathway has evolved into a multiplicity of alternative bifurcations and feedback mechanisms. The emerging signaling model includes the capacity of 5‐HT receptors GPCR superfamily to couple and initiate transduction through several G proteins depending on the tissue and/or the presence of interacting proteins. In turn, these G proteins may activate many effectors. On the other hand, the complexity increases further with the capacity of 5‐HT receptors to transduce signals through G‐protein‐independent pathways. These multiple couplings often participate in the control of gene transcription, thereby resulting in 5‐HT‐mediated long‐term (neuroplastic) adaptive functional changes. WIREs Membr Transp Signal 2012, 1:685–713. doi: 10.1002/wmts.50For further resources related to this article, please visit the WIREs website.

CAMP pathway and pituitary tumorigenesis

The pituitary is the target of different neurohormones that have a crucial role in the control of cell differentiation, cell proliferation and hormone secretion by recognizing specific receptors belonging to the G Protein-Coupled Receptor super-family (GPCR). Evidence from in vitro studies and naturally occurring human diseases indicate that several endocrine cells, and particularly somatotrophs, recognize cAMP as a growth factor. Accordingly, mutations of the alpha subunit of the stimulatory G protein gene (GNAS) leading to the constitutive activation of adenylyl cyclase (i.e. gsp oncogene) have been recognized in a significant proportion of GH-secreting pituitary adenomas. The role of cAMP in the control of cell proliferation in selected cell types and in particular in somatotroph cells has been further confirmed by identification of genetics defect affecting the regulatory subunit IA of PKA. The role of cAMP in the control of cell proliferation as well as the crosstalk with different intracellular signalling pathways will be discussed.

Membrane proteins, magic-angle spinning, and in-cell NMR

In PNAS, Renault et al. (1) describe an advance that will help reveal the atomic details of membrane proteins in living cells. Membrane proteins comprise one-third of all cellular proteins, including biologically crucial molecules such as ion channels, G protein-coupled receptors (GPCRs), and transporters. The GPCR superfamily alone comprises nearly half of our current drug targets. Despite their importance and prevalence, relatively few membrane protein structures are known. Of the more than 20,000 unique high-resolution protein structures in the Protein Data Bank (www.pdb.org) (2), less than 0.5% are membrane proteins. The paucity of membrane protein structures reflects difficulties in preparing sufficient quantities of protein for structural studies as well as technical challenges faced by both X-ray crystallography and NMR spectroscopy.