Structural Connection between Activation Microswitch and Allosteric Sodium Site in GPCR Signaling

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GRK2 Targeted Knock-down Results in Spontaneous Hypertension,and Altered Vascular GPCR Signaling

Hypertension, elevated arterial pressure, occurs as the consequence of increased peripheral resistance. G protein-coupled receptors (GPCRs) contribute to the regulation of vasodilator and vasoconstrictor responses, and their activity is regulated by a family of GPCR kinases (GRKs). GRK2 expression is increased in hypertension and this facilitates the development of the hypertensive state by increasing the desensitization of GPCRs important for vasodilation. We demonstrate here, that genetic knockdown of GRK2 using a small hairpin (sh) RNA results in altered vascular reactivity and the development of hypertension between 8–12 weeks of age in shGRK2 mice due to enhanced Gα_q/11 signaling. Vascular smooth muscle cells (VSMCs) cultured from shGRK2 knockdown mice show increases in GPCR-mediated Gα_s and Gα_q/11 signaling, as the consequence of reduced GRK2-mediated desensitization. In addition, agonists and biased agonists exhibited age-dependent alterations in ERK1/2 and Akt signaling, as well as cell proliferation and migration responses in shGRK2 knockdown VSMCs when cultured from mice that are either 3 months or 6 months of age. Changes in angiotensin II-stimulated ERK1/2 phosphorylation are observed in VSMCs derived from 6-week-old shGRK2 mice prior to the development of the hypertensive phenotype. Thus, our findings indicate that the balance between mechanisms regulating vascular tone are shifted to favor vasoconstriction in the absence of GRK2 expression and that this leads to the age-dependent development of hypertension, as a consequence of global alterations in GPCR signaling. Consequently, therapeutic strategies that target GRK2 activity, not expression, may be more effective for the treatment of hypertension.     

Interrogating the Spatiotemporal Landscape of Neuromodulatory GPCR Signaling by Real-Time Imaging of cAMP in Intact Neurons and Circuits

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Proton transfer-mediated GPCR activation

G-protein coupled receptors (GPCRs) play essential roles in signal transduction from the environment into the cell. While many structural features have been elucidated in great detail, a common functional mechanism on how the ligand-binding signal is converted into a conformational change on the cytoplasmic face resulting in subsequent activation of downstream effectors remain to be established. Based on available structural and functional data of the activation process in class-A GPCRs, we propose here that a change in protonation status, together with proton transfer via conserved structural elements located in the transmembrane region, are the key elements essential for signal transduction across the membrane.     

G Protein-coupled Receptor (GPCR) Signaling via Heterotrimeric G Proteins from Endosomes

Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a “second wave” of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors.     

Theme and variations on kinetics of GPCR activation/deactivation

G protein-coupled receptors (GPCRs) initiate intracellular signaling pathways in response to physiologically and medically important extracellular ligands such as peptide and large glycoprotein hormones, neurotransmitters, sensory stimuli (odorant and taste molecules, light), calcium, l-amino acids, and are the target of many clinical drugs. The conversion of these extracellular stimuli into intracellular signals involves sequential and reversible reactions that initially take place at the plasma membrane. These reactions are mediated not only by dynamic interactions between ligands, receptors and heterotrimeric G proteins, but also by conformational changes associated with the activation/deactivation process of each protein. This review discusses the kinetic characteristics and rate-limiting reactions engaged in signal propagation that are involved in systems as diverse as neurotransmitter and hormonal signaling, and that have been recorded in live cells by Förster resonance energy transfer (FRET) approaches.     

GPCR activation: protonation and membrane potential

GPCR proteins represent the largest family of signaling membrane proteins in eukaryotic cells. Their importance to basic cell biology, human diseases, and pharmaceutical interventions is well established. Many crystal structures of GPCR proteins have been reported in both active and inactive conformations. These data indicate that agonist binding alone is not suffi cient to trigger the conformational change of GPCRs necessary for binding of downstream G-proteins, yet other essential factors remain elusive. Based on analysis of available GPCR crystal structures, we identifi ed a potential conformational switch around the conserved Asp2.50, which consistently shows distinct conformations between inactive and active states. Combining the structural information with the current literature, we propose an energy-coupling mechanism, in which the interaction between a charge change of the GPCR protein and the membrane potential of the living cell plays a key role for GPCR activation.     

A hypothesis for GPCR activation

Growing evidence that rhodopsin (RD) and related G protein-coupled receptors form functional dimers/oligomers, followed by direct proof (using atomic force microscopy) that in the retina disc membrane RD associates into a paracrystalline network of rows of dimers, need models of the RD-transducin (Gt) complex that would envision an optimal RD dimer/oligomer able to satisfy all well-documented interactions with Gt. Of the models proposed so far, only a few refer to RD dimers and only one of them proposes a complex of Gt with an RD oligomer (Filipek S, Krzyko KA, Fotiadis D, Liang Y, Saperstein DA, Engel, A, Palczewski K Photochem Photobiol Sci 3: 628–638, 2004). This paper puts forward a hypothesis on another arrangement of RD monomers into the reported network of rows of dimers. Arguments for the compatibility of this set-up with interactions and activation of RD in the complex with Gt, in particular, with the well-documented movement of transmembrane helix 6 and cytosolic loop 3, which is vital for RD activation, are provided and discussed.This revised version was published online in June 2005 with corrections to the acknowledgements.     

Miniaturization of a Mammalian Cell-Based Assay: Luciferase Reporter Gene Readout in a 3 Microliter 1536-Well Plate

The combined efforts of the fields of combinatorial chemistry and genomics have significantly increased the number of compounds and therapeutic targets available for screening. The number of compounds will reach into the million range in the near future and provide vast chemical diversity for drug discovery. However, this reservoir of chemical diversity creates downstream hurdles for any screening effort. Properly examining this number of compounds increases investments dramatically, both in the number of dollars spent and amount of limited reagents depleted. Traditional HTS techniques, such as the use of 96-well microtiter plates, have paved the way for faster processing speeds, but are being rapidly overwhelmed by screening demands. Miniaturization of such assays will allow for greater throughput, while concurrently reducing cost. To date, miniaturization efforts have been most successfully applied to bacterial and soluble protein based assays. Questions about the ability to deliver microquantities of mammalian cells without disruption of the cell membrane and/or activation of stress responses have been raised. An assay has been developed in which a human T-cell screen has been adapted to a 1536-well plate format. Through the use of a luciferase reporter gene system, it is shown that a mammalian cell-based assay may be successfully performed in 3 μl and potent inhibitors of the target of interest identified.     

The Adhesion GPCR CD97/ADGRE5 inhibits apoptosis

The Adhesion G protein-coupled receptor (GPCR) CD97/ADGRE5 is induced, upregulated, and/or biochemically modified in various malignancies, compared to the corresponding normal tissues. As tumor cells are generally more resistant to apoptosis, we here studied the ability of CD97 to regulate tumor cell survival under apoptotic conditions. Stable overexpression of wild-type CD97 reduced serum starvation- and staurosporine-induced intrinsic and tumor necrosis factor (TNF)/cycloheximide-induced extrinsic apoptosis, indicated by an increase in cell viability, a lower percentage of cells within the subG0/G1 phase, expressing annexin V, or having condensed nuclei, and a reduction of DNA laddering. Protection from cell death by CD97 was accompanied by an inhibition of caspase activation and modulation of anti- and pro-apoptotic members of the BCL-2 superfamily. shRNA-mediated knockdown of CD97 and, in part, truncation of the seven-span transmembrane (TM7) region of CD97 increased caspase-mediated apoptosis. Protection from apoptosis required not only the TM7 region but also cleavage of the receptor at its GPCR proteolysis site (GPS), whereas alternative splicing of its extracellular domain had no effect. Together, our data indicate a role of CD97 in tumor cell survival.     

A constitutively active GPCR governs morphogenic transitions in Cryptococcus neoformans

Sex in fungi is driven by peptide pheromones sensed through seven‐transmembrane pheromone receptors. In Cryptococcus neoformans, sexual reproduction occurs through an outcrossing/heterothallic a ‐ sexual cycle or an inbreeding/homothallic – unisexual mating process. Pheromone receptors encoded by the mating‐type locus ( MAT ) mediate reciprocal pheromone sensing during opposite‐sex mating and contribute to but are not essential for unisexual mating. A pheromone receptor‐like gene, CPR2 , was discovered that is not encoded by MAT and whose expression is induced during a ‐ mating. cpr2 mutants are fertile but have a fusion defect and produce abnormal hyphal structures, whereas CPR2 overexpression elicits unisexual reproduction. When heterologously expressed in Saccharomyces cerevisiae , Cpr2 activates pheromone responses in the absence of any ligand. This constitutive activity results from an unconventional residue, Leu²²², in place of a conserved proline in transmembrane domain six; a Cpr2^L222P mutant is no longer constitutively active. Cpr2 engages the same G‐protein activated signalling cascade as the Ste3 a /α pheromone receptors, and thereby competes for pathway activation. This study established a new paradigm in which a naturally occurring constitutively active G protein‐coupled receptor governs morphogenesis in fungi.     

GPCR structures in drug design,emerging opportunities with new structures

In recent years, GPCR targets from diverse regions of phylogenetic space have been determined. This effort has culminated this year in the determination of representatives of all major classes of GPCRs (A, B, C, and F). Although much of the now well established knowledge on GPCR structures has been known for some years, the new high-resolution structures allow structural insight into the causes of ligand efficacy, biased signaling, and allosteric modulation. In this digest the structural basis for GPCR signaling in the light of the new structures is reviewed and the use of the new non-class A GPCRs for drug design is discussed.     

Relevance of rhodopsin studies for GPCR activation

Rhodopsin, the dim-light photoreceptor present in the rod cells of the retina, is both a retinal-binding protein and a G protein-coupled receptor (GPCR). Due to this conjunction, it benefits from an arsenal of spectroscopy techniques that can be used for its characterization, while being a model system for the important family of Class A (also referred to as “rhodopsin-like”) GPCRs. For instance, rhodopsin has been a crucial player in the field of GPCR structural biology. Until 2007, it was the only GPCR for which a high-resolution crystal structure was available, so all structure–activity analyses on GPCRs, from structure-based drug discovery to studies of structural changes upon activation, were based on rhodopsin. At present, about a third of currently available GPCR structures are still from rhodopsin. In this review, I show some examples of how these structures can still be used to gain insight into general aspects of GPCR activation. First, the analysis of the third intracellular loop in rhodopsin structures allows us to gain an understanding of the structural and dynamic properties of this region, which is absent (due to protein engineering or poor electron density) in most of the currently available GPCR structures. Second, a detailed analysis of the structure of the transmembrane domains in inactive, intermediate and active rhodopsin structures allows us to detect early conformational changes in the process of ligand-induced GPCR activation. Finally, the analysis of a conserved ligand-activated transmission switch in the transmembrane bundle of GPCRs in the context of the rhodopsin activation cycle, allows us to suggest that the structures of many of the currently available agonist-bound GPCRs may correspond to intermediate active states. While the focus in GPCR structural biology is inevitably moving away from rhodopsin, in other aspects rhodopsin is still at the forefront. For instance, the first studies of the structural basis of disease mutants in GPCRs, or the most detailed analysis of cellular GPCR signal transduction networks using a systems biology approach, have been carried out in rhodopsin. Finally, due again to its unique properties among GPCRs, rhodopsin will likely play an important role in the application of X-ray free electron laser crystallography to time-resolved structural biology in membrane proteins. Rhodopsin, thus, still remains relevant as a model system to study the molecular mechanisms of GPCR activation. This article is part of a Special Issue entitled: Retinal Proteins—You can teach an old dog new tricks.     

Evolving cryo-EM structural approaches for GPCR drug discovery

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Modulation of Glucagon-like Peptide-1 (GLP-1) Potency by Endocannabinoid-like Lipids Represents a Novel Mode of Regulating GLP-1 Receptor Signaling

Glucagon-like peptide-1 (GLP-1) analogs are approved for treatment of type 2 diabetes and are in clinical trials for disorders including neurodegenerative diseases. GLP-1 receptor (GLP-1R) is expressed in many peripheral and neuronal tissues and is activated by circulating GLP-1. Other than food intake, little is known about factors regulating GLP-1 secretion. Given a normally basal circulating level of GLP-1, knowledge of mechanisms regulating GLP-1R signaling, which has diverse functions in extrapancreatic tissues, remains elusive. In this study, we found that the potency of GLP-1, not exendin 4, is specifically enhanced by the endocannabinoid-like lipids oleoylethanolamide (OEA) and 2-oleoylglycerol but not by stearoylethanolamide (SEA) or palmitoylethanolamide. 9.2 μm OEA enhances the potency of GLP-1 in stimulating cAMP production by 10-fold but does not affect its receptor binding affinity. OEA and 2-oleoylglycerol, but not SEA, bind to GLP-1 in a dose-dependent and saturable manner. OEA but not SEA promoted GLP-1(7–36) amide to trypsin inactivation in a dose-dependent and saturable manner. Susceptibility of GLP-1(7–36) amide to trypsin inactivation is increased 40-fold upon binding to OEA but not to SEA. Our findings indicate that OEA binds to GLP-1(7–36) amide and enhances the potency that may result from a conformational change of the peptide. In conclusion, modulating potency of GLP-1 by physiologically regulated endocannabinoid-like lipids allows GLP-1R signaling to be regulated spatiotemporally at a constant basal GLP-1 level.     

A single chemosensory GPCR is required for a concentration-dependent behavioral switching in C. elegans

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A model for the evaluation of domain based classification of GPCR

G-Protein Coupled Receptors (GPCR) are the largest family of membrane bound receptor and plays a vital role in various biological processes with their amenability to drug intervention. They are the spotlight for the pharmaceutical industry. Experimental methods are both time consuming and expensive so there is need to develop a computational approach for classification to expedite the drug discovery process. In the present study domain based classification model has been developed by employing and evaluating various machine learning approaches like Bagging, J48, Bayes net, and Naive Bayes. Various softwares are available for predicting domains. The result and accuracy of output for the same input varies for these software''s. Thus, there is dilemma in choosing any one of it. To address this problem, a simulation model has been developed using well known five softwares for domain prediction to explore the best predicted result with maximum accuracy. The classifier is developed for classification up to 3 levels for class A. An accuracy of 98.59% by Naïve Bayes for level I, 92.07% by J48 for level II and 82.14% by Bagging for level III has been achieved.