Modelling and simulation of biased agonism dynamics at a G protein-coupled receptor.

Theoretical models of G protein-coupled receptor (GPCR) concentration-response relationships often assume an agonist producing a single functional response via a single active state of the receptor. These models have largely been analysed assuming steady-state conditions. There is now much experimental evidence to suggest that many GPCRs can exist in multiple receptor conformations and elicit numerous functional responses, with ligands having the potential to activate different signalling pathways to varying extents-a concept referred to as biased agonism, functional selectivity or pluri-dimensional efficacy. Moreover, recent experimental results indicate a clear possibility for time-dependent bias, whereby an agonist's bias with respect to different pathways may vary dynamically. Efforts towards understanding the implications of temporal bias by characterising and quantifying ligand effects on multiple pathways will clearly be aided by extending current equilibrium binding and biased activation models to include G protein activation dynamics. Here, we present a new model of time-dependent biased agonism, based on ordinary differential equations for multiple cubic ternary complex activation models with G protein cycle dynamics. This model allows simulation and analysis of multi-pathway activation bias dynamics at a single receptor for the first time, at the level of active G protein (αGTP), towards the analysis of dynamic functional responses. The model is generally applicable to systems with NG G proteins and N* active receptor states. Numerical simulations for NG=N*=2 reveal new insights into the effects of system parameters (including cooperativities, and ligand and receptor concentrations) on bias dynamics, highlighting new phenomena including the dynamic inter-conversion of bias direction. Further, we fit this model to 'wet' experimental data for two competing G proteins (Gi and Gs) that become activated upon stimulation of the adenosine A1 receptor with adenosine derivative compounds. Finally, we show that our model can qualitatively describe the temporal dynamics of this competing G protein activation.

Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast.

BACKGROUND AND PURPOSE: The glucagon-like peptide 1 (GLP-1) receptor performs an important role in glycaemic control, stimulating the release of insulin. It is an attractive target for treating type 2 diabetes. Recently, several reports of adverse side effects following prolonged use of GLP-1 receptor therapies have emerged: most likely due to an incomplete understanding of signalling complexities. EXPERIMENTAL APPROACH: We describe the expression of the GLP-1 receptor in a panel of modified yeast strains that couple receptor activation to cell growth via single Gα/yeast chimeras. This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity. KEY RESULTS: The GLP-1 receptor functionally coupled to the chimeras representing the human Gαs, Gαi and Gαq subunits. Calculation of the dissociation constant for a receptor antagonist, exendin-3 revealed no significant difference between the two systems. We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the Gαi pathway. We extended the use of the system to investigate small-molecule allosteric compounds and the closely related glucagon receptor. CONCLUSIONS AND IMPLICATIONS: These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future.

Interaction of PLD1b with actin in antigen-stimulated mast cells.

Phosphatidic acid, the product of phospholipase D catalysed phosphatidylcholine hydrolysis is an important signalling molecule that has been implicated in regulation of actin cytoskeleton remodelling and secretion from mast cells. We show that human PLD1b (hPLD1b) is an actin-binding protein and the N-terminus is predominantly involved in this interaction. Protein kinase C (PKC) is a major upstream regulator of PLD activity and PKC phosphorylation sites have been identified within the N-terminus of PLD1b at serine 2 and threonine 147. Over-expression of wild type hPLD1b in mast cells showed that antigen stimulation significantly enhanced co-localisation of PLD1b with actin structures. Mutation of serine 2 to alanine abolished antigen-induced co-localisation whereas mutation of threonine 147 had less dramatic effects on co-localisation. The absence of co-localisation of PLD1b (S2A) with actin coincides with a significant decrease in PLD activity in cells expressing the PLD1b (S2A) mutant. In resting RBL-2H3 cells, mutation of serine 2 to aspartate resulted in constitutive co-localisation of PLD with the actin cytoskeleton, coincident with restored PLD activity. These results reveal that serine 2 is an important regulatory site involved in controlling PLD enzyme activity and the interaction between PLD and actin.

Corticotropin-releasing hormone receptors.

Corticotropin-releasing hormone (CRH) and related peptides (urocortins, sauvagine, urotensin) play a central role in the co-ordination of autonomic, behavioural, cardiovascular, immune and endocrine responses to stressful stimuli. Their actions are mediated through activation of two types of G-protein-coupled receptors encoded by separate genes. In this review we focus on the diverse structural and functional characteristics of the family of CRH-like peptides and their receptors.

Identification of proteases with shared functions to the proprotein processing protease Krp1 in the fission yeast Schizosaccharomyces pombe.

Many secretory proteins are synthesized as inactive proproteins that undergo proteolytic activation as they travel through the eukaryotic secretory pathway. The best characterized family of processing enzymes are the prohormone convertases or kexins, and these are responsible for the processing of a wide variety of prohormones and other precursors. Recent work has identified other proteases that appear to be involved in proprotein processing, but characterization of these enzymes is at an early stage. Krp1 is the only kexin identified in the fission yeast Schizosaccharomyces pombe, in which it is essential for cell viability. We have used a genetic screen to identify four proteases with specificities that overlap Krp1. Two are serine proteases, one is a zinc metalloprotease (glycoprotease) and one is an aspartyl protease that belongs to the recently described yapsin family of processing enzymes. All four proteases support the growth of a yeast strain lacking Krp1, and each is able to process the P-factor precursor, the only substrate currently known to be processed by Krp1.

A temperature-sensitive Krp1 allows in vivo characterization of kexin activation.

Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes are themselves made as inactive precursors and we have investigated the activation of Krp1, a kexin from the fission yeast Schizosaccharomyces pombe. As Krp1 is essential for cell growth, we have used a krp1ts strain to investigate the role of the prosequence in the activation process. Mutations that reduce either the efficiency with which the prosequence is released or the rate at which the released prosegment is subsequently cleaved at an internal site are less active when assayed in vivo. We also show that prosegments lacking an internal dibasic motif can act as autoinhibitors and prevent activation of the catalytic fragment. Krp1 constructs containing prosequences based on these inhibitors do not become active in vitro. Surprisingly, the same constructs do become active in the intact cell and appear to suggest that alternative activation processes can be used by these enzymes.

Sxa2 is a serine carboxypeptidase that degrades extracellular P-factor in the fission yeast Schizosaccharomyces pombe.

Stimulating the fission yeast Schizosaccharomyces pombe with mating pheromones brings about responses that lead to cell conjugation. Persistent stimulation does not, however, induce a continuous response as the cells become desensitized to the presence of the pheromone. One mechanism that contributes to desensitization in M-cells is the release of a carboxypeptidase that inactivates the extracellular P-factor pheromone. Production of the carboxypeptidase requires a functional sxa2 gene. In this study, we report the first molecular characterization of the Sxa2 protein and provide direct evidence that it is the carboxypeptidase that degrades P-factor. Sxa2 is synthesized as a precursor that undergoes an internal cleavage event catalysed by a protease with specificity for basic residues. This generates a series of catalytically active N-terminal fragments and an inactive C-terminal fragment. Cleavage is essential for activation of the carboxypeptidase and, although the C-terminal fragment is inactive, it is required for the N-terminal fragment to attain activity.

Extracellular degradation of agonists as an adaptive mechanism.

Many cellular responses are initiated by the binding of an extracellular ligand to receptors at the cell surface. The removal of these ligands, or agonists, would therefore be expected to contribute to the process by which cells recover from stimulation. While dilution in the extracellular medium reduces the concentration of most agonists, many cells have developed specific mechanisms for removing particular ligands. One of the more effective mechanisms is to degrade the extracellular agonist and here we review the production and action of the enzymes responsible for the degradation of a number of these agonists.

The processing of yeast pheromones.

Yeast provides an attractive system in which to study proprotein processing. Many of their processing events are remarkably similar to those in higher eukaryotes and their amenability to experimental manipulation permits approaches that are not always feasible in multicellular organisms. Analysing the biosynthesis of the yeast mating pheromones has been particularly rewarding and has provided insights into both the proteolytic aspects of processing and other post-translational events such as prenylation and carboxymethylation.

The sxa2-dependent inactivation of the P-factor mating pheromone in the fission yeast Schizosaccharomyces pombe.

Haploid cells of the fission yeast Schizosaccharomyces pombe exist in one of two mating types, referred to as M and P. Conjugation occurs between cells of opposite mating type and is controlled by the reciprocal action of diffusible pheromones. Loss of function of the sxa2 gene in M cells causes hypersensitivity to the P-factor mating pheromone and a reduction in mating efficiency. Here we demonstrate the secretion of an sxa2-dependent carboxypeptidase that inactivates P-factor by removal of the C-terminal leucine residue.

Automated liquid chromatographic method for the determination of paracetamol and six metabolites in human urine.

An automated liquid chromatographic method, with a coefficient of variation for total imprecision of less than 4%, has been developed for the quantitative determination of paracetamol, paracetamol-4-glucuronide, paracetamol-4-sulphate, paracetamol-3-cysteine, paracetamol-3-mercapturate, 3-hydroxyparacetamol-3-sulphate and 3-methoxyparacetamol-4-sulphate in urine samples. The gradient elution system was based on 0.067 M phosphate buffer (pH 2.0) and acetonitrile on an octadecylsilica column. The on-column detection limit using an ultraviolet detector at 254 nm for each of the compounds using 3-hydroxyacetanilide as internal standard was of the order of 10-50 ng from urine and 2-10 ng from water. Application of the method to 24-h urine samples from subjects who had received a therapeutic dose of the drug confirmed the findings of previous studies for the importance of the glucuronide, sulphate, mercapturate and cysteine conjugates. 3-Hydroxyparacetamol-3-sulphate was shown to be present in the urine of all volunteers and to account for up to 5% of the dose. 3-Methoxyparacetamol-4-sulphate was not detected in any urine samples and if present as a metabolite must account for less than 0.1% of the dose.

A model of focal cortical contusion in gerbils.

An experimental model of focal laceration and contusion in gerbils is described. Associated with this injury are systemic changes which are neurogenically mediated and result in an immediate reduction in blood pressure, bradycardia, and generalized reduction in cerebral blood flow. There is generalized edema, as judged by a decreased specific gravity in the brain, probably related to reduced blood flow; superimposed on this, there is an edema gradient which is maximal close to the injury. This, in turn, affects the local capillary bed and prevents any local increase in flow. A separate group studied over a longer time period (6 hours) did not reveal egress of Evans blue into the surrounding tissue and this is in contrast to reports from cold-injury studies.

Cerebrovascular effects of prostaglandin inhibitors in the gerbil.

Autoregulation of cerebral blood flow (CBF) to mean arterial blood pressure (MABP) of 40-50 mm Hg has been demonstrated in the spontaneously breathing gerbil anaesthetised with barbiturate (60 mg/kg). CO2 reactivity has also been assessed at 2.8% change CBF/mm Hg change in arterial PCO2. In six animals pretreated with indomethacin (3 mg/kg), autoregulation was preserved although the resting CBF was significantly reduced, but CO2 reactivity was completely abolished. 1-n-Butyl imidazole, a specific thromboxane synthetase inhibitor, was used in six other animals (3 mg/kg), and this abolished CO2 reactivity while preserving autoregulation; the effect of this agent has not been described previously. Both drugs inhibit different pathways of prostaglandin metabolism and may interfere with normal CO2 reactivity in several ways. Two explanations are that prostaglandins constitute the final common pathway in effecting cerebrovascular response to CO2 or, alternatively, that the free radicals and ionic fluxes generated during prostaglandin metabolism are a coincidental source of the hydrogen ion changes required.

Are prostaglandins involved in experimental ischemic edema in gerbils?

Sixty-five male gerbils, divided into 3 groups, were used in this study, in which focal brain specific gravity, taken as a measure of edema, was compared to the corresponding focal cerebral blood flow using the hydrogen washout technique. Extracranial unilateral or bilateral carotid ligation was performed and one hour later the animal was sacrificed. When focal blood flow was less than 20 ml/100 g/min, edema developed and increased with progressive ischemia, reaching maximal values at 5-7 ml/100 g/min. In the zero flow situation there was no edema. Pretreatment of the other 2 groups with indomethacin or dexamethasone, did not prevent edema formation at flows of 20-12 ml/200 g/min, but considerably reduced the edema previously noted at low flows (5-7 ml/100 g/min). The drugs did not affect the decreased flow in the ischemic area. We conclude that prostaglandins, released by membrane disruption, are involved in the development of ischemic edema.