Analyzing kinetic signaling data for G-protein-coupled receptors

In classical pharmacology, bioassay data are fit to general equations (e.g. the dose response equation) to determine empirical drug parameters (e.g. EC50 and Emax), which are then used to calculate chemical parameters such as affinity and efficacy. Here we used a similar approach for kinetic, time course signaling data, to allow empirical and chemical definition of signaling by G-protein-coupled receptors in kinetic terms. Experimental data are analyzed using general time course equations (model-free approach) and mechanistic model equations (mechanistic approach) in the commonly-used curve-fitting program, GraphPad Prism. A literature survey indicated signaling time course data usually conform to one of four curve shapes: the straight line, association exponential curve, rise-and-fall to zero curve, and rise-and-fall to steady-state curve. In the model-free approach, the initial rate of signaling is quantified and this is done by curve-fitting to the whole time course, avoiding the need to select the linear part of the curve. It is shown that the four shapes are consistent with a mechanistic model of signaling, based on enzyme kinetics, with the shape defined by the regulation of signaling mechanisms (e.g. receptor desensitization, signal degradation). Signaling efficacy is the initial rate of signaling by agonist-occupied receptor (kτ), simply the rate of signal generation before it becomes affected by regulation mechanisms, measurable using the model-free analysis. Regulation of signaling parameters such as the receptor desensitization rate constant can be estimated if the mechanism is known. This study extends the empirical and mechanistic approach used in classical pharmacology to kinetic signaling data, facilitating optimization of new therapeutics in kinetic terms

NanoBiT ‐ and NanoBiT/BRET ‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model

Background and Purpose Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD$_{7}$, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. Experimental Approach SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD$_{7}$, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD$_{7}$ using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. Key Results With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD$_{7}$ was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD$_{7}$ obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. Conclusions and Implications Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD$_{7}$ binding should be performed using receptors expressed under endogenous promotion

Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects

Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects

Anthropometric and Physical Qualities of Elite Male Youth Rugby League Players

Rugby league is a collision team sport played at junior and senior levels worldwide, whereby players require highly developed anthropometric and physical qualities (i.e., speed, change of direction speed, aerobic capacity, muscular strength and power). Within junior levels, professional clubs and national governing bodies implement talent identification and development programmes to support the development of youth (i.e., 13-20 years) rugby league players into professional athletes. This review presents and critically appraises the anthropometric and physical qualities of elite male youth rugby league players aged between 13 and 20 years by age category, playing standard and playing position. Height, body mass, body composition, linear speed, change of direction speed, aerobic capacity, muscular strength and power characteristics are presented and demonstrate that qualities develop with age and differentiate between playing standard and playing position. This highlights the importance of anthropometric and physical qualities for the identification and development of youth rugby league players. However, factors such as maturity status, variability in development, longitudinal monitoring and career attainment should be considered to help understand, identify and develop the physical qualities of youth players. Further extensive research is required into the anthropometric and physical qualities of youth rugby league players, specifically considering national standardized testing batteries, links between physical qualities and match performance, together with intervention studies, to inform the physical development of youth rugby league players for talent identification and development purposes

Gene Ontology Consortium: going forward

The Gene Ontology (GO; http://www.geneontology.org) is a community-based bioinformatics resource that supplies information about gene product function using ontologies to represent biological knowledge. Here we describe improvements and expansions to several branches of the ontology, as well as updates that have allowed us to more efficiently disseminate the GO and capture feedback from the research community. The Gene Ontology Consortium (GOC) has expanded areas of the ontology such as cilia-related terms, cell-cycle terms and multicellular organism processes. We have also implemented new tools for generating ontology terms based on a set of logical rules making use of templates, and we have made efforts to increase our use of logical definitions. The GOC has a new and improved web site summarizing new developments and documentation, serving as a portal to GO data. Users can perform GO enrichment analysis, and search the GO for terms, annotations to gene products, and associated metadata across multiple species using the all-new AmiGO 2 browser. We encourage and welcome the input of the research community in all biological areas in our continued effort to improve the Gene Ontology

Modelling the activation of G-protein coupled receptors by a single drug

In this paper, the most popular proposed mechanism for activation of G-protein coupled receptors (GPCRs) - the shuttling mechanism - is modelled mathematically. An asymptotic analysis of this model clarifies the dynamics of the system in the presence of a drug, in particular identifying which reactions dominate during the different timescales. The modelling also reveals challenging behaviour in the form of a peak response. This new mechanism gives simple explanations for complex, possibly misunderstood, behaviour. © 2009 Elsevier Inc. All rights reserved

Mathematical modelling of signalling in a two-ligand G-protein coupled receptor system: Agonist-antagonist competition

A new mathematical model of cell signalling for a two-ligand G-protein coupled receptor (GPCR) system is presented. This model extends the single-ligand cubic ternary complex to account for the possibility of an agonist and an antagonist competing for receptor sites. The G-protein cycle is included, and signalling as far as the dissociated Gα subunit is considered. Numerical simulations are performed, and the effects on the system dynamics, such as peak and plateau behaviour, of antagonist "stickiness", and of the doses of agonist and antagonist, are discussed. Under certain parameter regimes, the plateau response is subject to surmountable antagonism, while the peak response is subject to insurmountable antagonism. The numerical results reveal responses evolving over a number of time-scales. An asymptotic analysis is presented which identifies dominant reactions and gives leading order solutions over these various time-scales, for a number of parameter regimes. © 2009 Elsevier Inc. All rights reserved

Modelling of the activation of G-protein coupled receptors: drug free constitutive receptor activity

G-protein coupled receptors (GPCRs) form a crucial component of approximately 80% of hormone pathways. In this paper, the most popular mechanism for activation of GPCRs-the shuttling mechanism-is modelled mathematically. An asymptotic analysis of this model clarifies the dynamics of the system in the absence of drug, in particular which reactions dominate during the different timescales. Equilibrium analysis of the model demonstrates the model's ability to predict constitutive receptor activity. © Springer-Verlag 2009