Function Of GPCRs Research Articles

Bradykinin receptor expression and bradykinin-mediated sensitization of human sensory neurons.

Bradykinin is a peptide implicated in inflammatory pain in both humans and rodents. In rodent sensory neurons, activation of B1 and B2 bradykinin receptors induces neuronal hyperexcitability. Recent evidence suggests that human and rodent dorsal root ganglia (DRG), which contain the cell bodies of sensory neurons, differ in the expression and function of key GPCRs and ion channels; whether bradykinin receptor expression and function are conserved across species has not been studied in depth. In this study, we used human DRG tissue from organ donors to provide a detailed characterization of bradykinin receptor expression and bradykinin-induced changes in the excitability of human sensory neurons. We found that B2 and, to a lesser extent, B1 receptors are expressed by human DRG neurons and satellite glial cells. B2 receptors were enriched in the nociceptor subpopulation. Using patch-clamp electrophysiology, we found that acute bradykinin increases the excitability of human sensory neurons, whereas prolonged exposure to bradykinin decreases neuronal excitability in a subpopulation of human DRG neurons. Finally, our analyses suggest that donor's history of chronic pain and age may be predictors of higher B1 receptor expression in human DRG neurons. Together, these results indicate that acute bradykinin-induced hyperexcitability, first identified in rodents, is conserved in humans and provide further evidence supporting bradykinin signaling as a potential therapeutic target for treating pain in humans.

New Insights into the Structure and Function of Class B1 GPCRs.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. Class B1 GPCRs constitute a subfamily of 15 receptors that characteristically contain large extracellular domains (ECDs) and respond to long polypeptide hormones. Class B1 GPCRs are critical regulators of homeostasis, and, as such, many are important drug targets. While most transmembrane proteins, including GPCRs, are recalcitrant to crystallization, recent advances in cryo-electron microscopy (cryo-EM) have facilitated a rapid expansion of the structural understanding of membrane proteins. As a testament to this success, structures for all the class B1 receptors bound to G proteins have been determined by cryo-EM in the past 5 years. Further advances in cryo-EM have uncovered dynamics of these receptors, ligands, and signaling partners. Here, we examine the recent structural underpinnings of the class B1 GPCRs with an emphasis on structure-function relationships.

GPCRs Are Optimal Regulators of Complex Biological Systems and Orchestrate the Interface between Health and Disease.

GPCRs arguably represent the most effective current therapeutic targets for a plethora of diseases. GPCRs also possess a pivotal role in the regulation of the physiological balance between healthy and pathological conditions; thus, their importance in systems biology cannot be underestimated. The molecular diversity of GPCR signaling systems is likely to be closely associated with disease-associated changes in organismal tissue complexity and compartmentalization, thus enabling a nuanced GPCR-based capacity to interdict multiple disease pathomechanisms at a systemic level. GPCRs have been long considered as controllers of communication between tissues and cells. This communication involves the ligand-mediated control of cell surface receptors that then direct their stimuli to impact cell physiology. Given the tremendous success of GPCRs as therapeutic targets, considerable focus has been placed on the ability of these therapeutics to modulate diseases by acting at cell surface receptors. In the past decade, however, attention has focused upon how stable multiprotein GPCR superstructures, termed receptorsomes, both at the cell surface membrane and in the intracellular domain dictate and condition long-term GPCR activities associated with the regulation of protein expression patterns, cellular stress responses and DNA integrity management. The ability of these receptorsomes (often in the absence of typical cell surface ligands) to control complex cellular activities implicates them as key controllers of the functional balance between health and disease. A greater understanding of this function of GPCRs is likely to significantly augment our ability to further employ these proteins in a multitude of diseases.

Structure, function and pharmacology of human itch GPCRs.

The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently1. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions2-5. MRGPRX2 couples to both Gi and Gq in mast cells6. Here we describe agonist-stabilized structures of MRGPRX2 coupled to Gi1 and Gq in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and Gq. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.

Alzheimer’s disease as a condition of accelerated aging of cerebrovascular endothelial function

Currently ~5.8 million Americans have Alzheimer’s disease (AD), whereby >95% of patients are ≥65 years old. Age‐related cognitive disorders including AD are associated with impaired blood flow delivery of oxygen and nutrients throughout the brain. Cerebrovascular endothelium is central to coordinating vasoreactivity of blood vessel networks for optimal cerebral blood flow. Thus we tested the hypothesis that cerebrovascular endothelial Gq‐protein‐coupled receptor (GPCR; P2Y) and K+ channel (KCa2.3/SK3 & KCa3.1/IK1, KIR2.1) function declines during progressive age and AD pathology. Using Fura‐2 photometry (intracellular Ca2+) and sharp electrodes (membrane potential), we measured the function of endothelial tubes of posterior cerebral arteries of male and female mice (n≥5/group): C57BL/6 [young (4–6 mo), middle‐aged (12–16 mo), and old (24–28 mo)] & 3xTgAD [young, no pathology (1–2 mo), mild cognitive impairment (MCI; 4–5 mo), extracellular Aβ plaques (Aβ; 6–8 mo), and Aβ plaques + neurofibrillary tangles (AβT; 12–15 mo)]. During normal aging, P2Y function in response to ATP (100 μM) was reduced in male vs. females during old age. In contrast, SKCa/IKCa channel function in response to NS309 (0.3–1 μM) decreased in old females vs. young females & old males. Activation of KIR channels to elevated extracellular K+ (15 mM KCl) decreased with advancing age regardless of sex. For 3xTgAD animals, P2Y function was maintained throughout AD pathology in females but decreased during AβT in males. SKCa/IKCa channel function was reduced in Aβ females vs. Aβ males. Further, KIR function decreased during Aβ and AβT groups regardless of sex. Altogether, AD pathology accelerates the impact of advancing age on cerebrovascular endothelium by at least 12 mo while indicating a sex‐based contrast in declined function of GPCRs and K+ channels across males and females respectively. Modulation of endothelial K+ channel activity may optimize cerebral blood flow during aging and AD to prevent and treat neurodegenerative disease.Support or Funding InformationThis research was supported by National Institutes of Health grants R00AG047198 & R56AG062169 (EJB).

A Discrete Presynaptic Vesicle Cycle for Neuromodulator Receptors

A major function of GPCRs is to inhibit presynaptic neurotransmitter release, requiring ligand-activated receptors to couple locally to effectors at terminals. The current understanding of how this is achieved is through receptor immobilization on the terminal surface. Here, we show that opioid peptide receptors, GPCRs that mediate highly sensitive presynaptic inhibition, are instead dynamic in axons. Opioid receptors diffuse rapidly throughout the axon surface and internalize after ligand-induced activation specifically at presynaptic terminals. We delineate a parallel regulated endocytic cycle for GPCRs operating at the presynapse, separately from the synaptic vesicle cycle, which clears activated receptors from the surface of terminals and locally reinserts them to maintain the diffusible surface pool. We propose an alternate strategy for achieving local control of presynaptic effectors that, opposite to using receptor immobilization and enforced proximity, is based on lateral mobility of receptors and leverages the inherent allostery of GPCR-effector coupling.

Structure and function of adhesion GPCRs and their ligands

Structure and function of adhesion GPCRs and their ligands

Abstract P277: Ceramide Modulates GPCR And TK Receptor Signaling To Impact Endothelial Functions And Blood Pressure

Ceramides are sphingolipids that modulate a variety of cellular processes, via two major modes of actions: by functioning as second messengers and by regulating the formation of lipid raft, important signaling platform. Receptor-mediated signaling orchestrates multiple pathophysiological processes, hence more than 50% of drugs clinically prescribed target mainly GPCRs. Alterations of ceramide levels are implicated in endothelial dysfunction, a key event in many cardiovascular diseases, including hypertension and atherosclerosis. However, specific molecular mechanisms of how ceramides modulate the structure and the function of GPCRs and TK receptors to impact endothelial functions remains unknown and unexplored. Thus, we generated mice lacking the endothelial sphingolipid de novo biosynthesis, by deleting the serine palmitoyltransferase long subunit 2, of the first enzyme of the pathway, named ECKO-Sptlc2. Systolic blood pressure was markedly increased in ECKO-Sptlc2 vs. control mice (122.1± 1.9 vs. 103.6±0.7 mmHg, n=9). Vasodilation of mesenteric arteries to acetylcholine and histamine (Gq-coupled receptors) and the NO-signaling downstream was preserved suggesting that altered membrane sphingolipid levels did not affect Gq-coupled receptor activation. On the contrary, vasodilation induced by the activation of tyrosine kinase receptors, i.e. VEGFR2 (Emax 32.8±3.5 vs. 55.7±3.6 % vasodilation, n=5), Gi-coupled GPCRs, i.e. S1PR1 (Emax 9.0±11.0 vs. 58.7 ±3.5 % vasodilation, n=5) and flow were markedly reduced. C16-, C24- and C24:1-ceramide are the most abundant ceramides in the endothelium and were markedly reduced by the loss of Sptlc2. Interestingly, the treatment of the mice and endothelial cells in culture with C16-, C24- and C24:1-ceramides, showed that C16-ceramide play a specific role in VEGFR2, IR, S1PR1 and flow mediated vasodilation. Mechanistically, C16-ceramide modulates VEGFR2 phosphorylation and downstream signaling in a concentration-dependent manner. The finding that C16-ceramide affects specific the function of Gi-coupled, TK but not Gq-coupled receptor is of great significance considering that alterations in C16-ceramide strongly correlate with CV conditions, such as CAD and heart failure.

Pharmacological Assays for Investigating the NOP Receptor.

The nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) is a G protein-coupled receptor involved in the regulation of several physiological functions and pathological conditions. Thus, researchers from academia and industry are pursuing NOP to discover and study novel pharmacological entities. In a multidisciplinary effort of pharmacologists, medicinal chemists, and molecular and structural biologists the mechanisms of NOP activation and inhibition have been, at least partially, disentangled. Here, we review the in vitro methodologies employed, which have contributed to our understanding of this target. We hope this chapter guides the reader through the mostly established assay platforms to investigate NOP pharmacology, and gives some hints taking advantage from what has already illuminated the function of other GPCRs. We analyzed the pharmacological results obtained with a large panel of NOP ligands investigated in several assays including receptor binding, stimulation of GTPγS binding, decrease of cAMP levels, calcium flux stimulation via chimeric G proteins, NOP/G protein and NOP/β-arrestin interaction, label-free assays such as dynamic mass redistribution, and bioassays such as the electrically stimulated mouse vas deferens.

Insights into the function of opioid receptors from molecular dynamics simulations of available crystal structures.

This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

Sweetness-induced activation of membrane dipole potential in STC-1 taste cells

The biological functions of cell membranes strongly influence the binding and transport of molecular species. We developed STC-1 cell line stably expressing the sweet taste receptor (T1R2/T1R3), and explored the possible correlation between sweeteners and membrane dipole potential of STC-1 cells. In this study, sweetener-induced dipole potential activation was elucidated using a fluorescence-based measurement technique, by monitoring the voltage sensitive probe Di-8-ANEPPS using a dual wavelength ratiometric approach. It indicated that the presence of sweeteners resulted in cell membrane dipole potential change, and interaction of artificial sweeteners with taste cells resulted in a greater reduction in potential compared with natural sweeteners. Our work presents a newly developed approach using a fluorescence-based measurement technique to study sweetener-induced dipole potential activation of STC-1 cells. This new approach could be used as a complementary tool to study the function of sweet taste receptors or other GPCRs and helps to understand the basis sweetness mechanism.

Identifying ligands at orphan GPCRs: current status using structure-based approaches.

GPCRs are the most successful pharmaceutical targets in history. Nevertheless, the pharmacology of many GPCRs remains inaccessible as their endogenous or exogenous modulators have not been discovered. Tools that explore the physiological functions and pharmacological potential of these 'orphan' GPCRs, whether they are endogenous and/or surrogate ligands, are therefore of paramount importance. Rates of receptor deorphanization determined by traditional reverse pharmacology methods have slowed, indicating a need for the development of more sophisticated and efficient ligand screening approaches. Here, we discuss the use of structure-based ligand discovery approaches to identify small molecule modulators for exploring the function of orphan GPCRs. These studies have been buoyed by the growing number of GPCR crystal structures solved in the past decade, providing a broad range of template structures for homology modelling of orphans. This review discusses the methods used to establish the appropriate signalling assays to test orphan receptor activity and provides current examples of structure-based methods used to identify ligands of orphan GPCRs. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Applications of yeast-based signaling sensor for characterization of antagonist and analysis of site-directed mutants of the human serotonin 1A receptor.

The monoamine neurotransmitter serotonin (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. One such receptor, the 5-HT1A receptor (HTR1A), is the most widely studied subtype and represents a significant molecular target in medicinal and therapeutic fields. Yeast-based fluorescent reporter systems have proven to be especially useful for GPCR assays, since detection using a fluorescent reporter considerably simplifies measurement procedures. However, previously reported systems using enhanced green fluorescent protein (EGFP) as the reporter in yeast still showed low signal-to-noise (S/N) ratios, making EGFP difficult to apply as an easily accessible tool. Therefore, we constructed a refined yeast-based GPCR biosensor employing a high-sensitivity strain that incorporated both a Gα-engineered receptor and a fluorescent reporter (ZsGreen). As we report here, the refined yeast-based fluorescent biosensor was applied successfully to antagonist characterization and analysis of site-directed mutants of the HTR1A receptor. Pindolol, a known antagonist of HTR1A, specifically inhibited agonist-induced signaling, demonstrating the ease of evaluating inhibition effects using our reporter strain. Characterization of site-specific receptor mutants confirmed the role of specific targeted residues, including the highly conserved DRY motif, in the activation of HTR1A. Thus, our refined yeast biosensor strain, which incorporates a ZsGreen reporter and an engineered Gα receptor, is expected to serve as a simple and practical sensing tool for evaluating the ligand candidates and defining residues important to the function of human GPCRs. Biotechnol.

Role of G-protein-coupled Receptor-related Genes in Insecticide Resistance of the Mosquito, Culex quinquefasciatus

G-protein-coupled receptors regulate signal transduction pathways and play diverse and pivotal roles in the physiology of insects, however, the precise function of GPCRs in insecticide resistance remains unclear. Using quantitative RT-PCR and functional genomic methods, we, for the first time, explored the function of GPCRs and GPCR-related genes in insecticide resistance of mosquitoes, Culex quinquefasciatus. A comparison of the expression of 115 GPCR-related genes at a whole genome level between resistant and susceptible Culex mosquitoes identified one and three GPCR-related genes that were up-regulated in highly resistant Culex mosquito strains, HAmCqG8 and MAmCqG6, respectively. To characterize the function of these up-regulated GPCR-related genes in resistance, the up-regulated GPCR-related genes were knockdown in HAmCqG8 and MAmCqG6 using RNAi technique. Knockdown of these four GPCR-related genes not only decreased resistance of the mosquitoes to permethrin but also repressed the expression of four insecticide resistance-related P450 genes, suggesting the role of GPCR-related genes in resistance is involved in the regulation of resistance P450 gene expression. This results help in understanding of molecular regulation of resistance development in Cx. quinquefasciatus.

Evolving pharmacology of orphan GPCRs: IUPHAR Commentary

The award of the 2012 Nobel Prize in Chemistry to Robert Lefkowitz and Brian Kobilka for their work on the structure and function of GPCRs, spanning a period of more than 20 years from the cloning of the human β2 -adrenoceptor to determining the crystal structure of the same protein, has earned both researchers a much deserved place in the pantheon of major scientific discoveries. GPCRs comprise one of the largest families of proteins, controlling many major physiological processes and have been a major focus of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) since its inception in 1987. We report here recent efforts by the British Pharmacological Society and NC-IUPHAR to define the endogenous ligands of 'orphan' GPCRs and to place authoritative and accessible information about these crucial therapeutic targets online.

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans. In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. We now report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the PKA-anchoring protein ezrin to facilitate PTH-responsive cAMP signaling events. Activation of ezrin-anchored PKA initiates NHERF1 phosphorylation to disassemble the ternary complex, release Npt2a, and thereby inhibit phosphate transport. Loss-of-function mutations stabilize an inactive NHERF1 conformation that we show is refractory to PKA phosphorylation and impairs assembly of the ternary complex. Compensatory mutations introduced in mutant NHERF1 re-establish the integrity of the ternary complex to permit phosphorylation of NHERF1 and rescue PTH action. These findings offer new insights into a novel macromolecular mechanism for the physiological action of a critical ternary complex, where anchored PKA coordinates the assembly and turnover of the Npt2a-NHERF1-ezrin complex.

ロドプシンおよび関連受容体結晶解析の進展

Most eukaryotic organisms sense a majority of extracellular signals such as light, odorants and neurotransmitters via heptahelical membrane receptors called GPCRs, which are structurally distinct from microbial retinal proteins (photoreceptors) . After several years from the first high-resolution structure determination of a prototypical GPCR visual rhodopsin in its inactive state, there has been significant progress over the past few years in the X-ray crystallographic study on this large family of receptors. Although the number of available structure is still limited, some valuable insights are obtained with regard to the structure and function of rhodopsin-like GPCRs.

The Role of Internal Water Molecules in the Structure and Function of the Rhodopsin Family of G Protein‐Coupled Receptors

Membrane receptors coupled to guanine nucleotide-binding regulatory proteins (commonly known as G protein-coupled ACHTUNGTRENNUNGreceptors, GPCRs) constitute one of the most attractive pharmaceutical targets, as around 40% of clinically prescribed drugs and 25% of the top-selling drugs act at these receptors. GPCRs are receptors for sensory signals of external origin such as odors, pheromones, or tastes; and for endogenous signals such as neurotransmitters, (neuro)peptides, divalent cations, proteases, glycoprotein hormones, and purine ligands. Phylogenetic analyses of the human genome have permitted GPCR sequences to be classified into five main families: rhodopsin (class A or family 1), secretin (class B or family 2), glutamate (class C or family 3), adhesion, and frizzled/ taste2. Specialized databases of GPCRs can be found at http://www.gpcr.org/7tm, http://gris.ulb.ac.be/, and http:// www.iuphar-db.org. Due to the low natural abundance of GPCRs and the difficulty in producing and purifying recombinant protein, only one member of this family, rhodopsin, the photoreceptor protein of rod cells, has been crystallized so far. Five structural models of inactive rhodopsin are available at the Protein Data Bank, at resolutions of 2.8 A (PDB IDs: 1F88 and 1HZX), 2.65 A (1GZM), 2.6 A (1L9H), and 2.2 A (1U19). Structural models of rhodopsin photointermediates such as bathorhodopsin (2G87), lumirhodopsin (2HPY), metarhodopsin I, and a photoactivated deprotonated intermediate reminiscent of metarhodopsin II (2I37) are also available. Rhodopsin is formed by an extracellular N terminus of four b-strands, seven transmembrane helices (TM1 to TM7) connected by alternating intracellular (I1 to I3) and extracellular (E1 to E3) hydrophilic loops, a disulfide bridge between E2 and TM3, and a cytoplasmic C terminus containing an a-helix (Hx8) parallel to the cell membrane. Statistical analysis of the residues forming the TM helices of the rhodopsin family of GPCRs shows a large number of conserved sequence patterns; this suggests a common TM structure. Thus, the availability of the rhodopsin structure allows the use of homology modeling techniques to build three-dimensional models of other homologous GPCRs. The putative structural homology between rhodopsin and other GPCRs probably does not extend to the extracellular domain, since the extracellular N terminus and loop fragments are highly variable in length and amino acid content. The class A family of GPCRs contains highly conserved Pro residues in the middle of TMs 5 (P5.50, conserved in 77% of the sequences), 6 (P6.50, 100%), and 7 (P7.50, 96%; residues are identified by the generic numbering scheme of Ballesteros and Weinstein, which allows easy comparison among residues in the 7TM segments of different receptors). Pro residues are normally observed in the TM helices of membrane proteins where they usually induce a significant distortion named a “Pro-kink”. This break arises in order to avoid a steric clash between the pyrrolidine ring of the Pro side chain (at position i) and the carbonyl oxygen of the residue in the preceding turn (position i 4) and leads to a distortion of the helical structure. However, TM segments of rhodopsin, either with or without Pro residues in their sequence are far from being standard Pro-kinked or ideal helices, respectively. Their distortions are energetically stabilized through complementary intraand interhelical interactions involving polar side chains, backbone carbonyls, and, in some cases, specific structural and functional water molecules embedded in the TM bundle. Here we review the role of these water molecules in the structure and function of GPCRs and in building computergenerated homology models of class A GPCRs. We propose that water molecules present in the vicinity of highly conserved motifs are most likely present in the rhodopsin family of GPCRs, being another conserved structural element in the family.