Analyzing kinetic signaling data for G-protein-coupled receptors

Abstract

In classical pharmacology, bioassay data are fit to general equations (e.g. the dose response equation) to determine empirical drug parameters (e.g. EC 50 and E max ), 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 Gprotein-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 agonistoccupied receptor (k τ ), 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 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.

Circuit-specific enteric glia regulate intestinal motor neurocircuits

“…****P < 0.0001. intensity of 0.9 ± 0.1 ΔF/s in glia compared with 0.2 ± 0.1 ΔF/s in neurons, suggesting CNO evoked greater and faster changes in the intracellular calcium concentration in glia than neurons. Glial responses to CNO were a near perfect fit to classical GPCR response models, while neuronal responses showed a weaker correlation (CNO R-square = 0.6940 and 0.9661 for neurons and glia, respectively) (38), likely reflecting the direct effects of CNO on glia and subsequent effects on neurons. Subsequent glial responses to serial CNO application were lower likely due to the brief drug washout period.…”

“…Final data values were quantified as fold change in mean cellular fluorescence intensity relative to baseline florescence intensity (ΔF/F o ) within a given cell. An analysis of Ca 2+ response time-course kinetics was conducted according to the equation describing the rise and fall to baseline time-course profile (38). For population recruitment studies evaluating the proportions of neurons or glia activated by FTS, a positive response was defined as a peak fluorescence value >5 SDs above the average baseline Ca 2+ level measured within a given cell for a fixed recording duration.…”

Development of fluorescent peptide G protein‐coupled receptor activation biosensors for NanoBRET characterization of intracellular allosteric modulators

“…The effect of high sodium concentration, as a known negative allosteric modulator of class A GPCR conformational change to an active state, 40,41 demonstrated a predicted decrease in agonist potency, and enhanced the partial agonism (reduced Rmax) apparent for those ligands (salmeterol, salbutamol) with lower intrinsic efficacy. Notably, the assay enabled simple collection of kinetic TMR‐Gαs19cha18 recruitment data and analysis of agonist pharmacology using initial TMR‐Gαs19cha18 rates of association, 34 providing the opportunity to routinely monitor time dependent, as well as equilibrium agonist behavior in one plate format.…”

Prior Acoustic Trauma Alters Type II Afferent Activity in the Mouse Cochlea

“…Delayed responses occurring minutes after the initial tissue damage in type II afferents and epithelial cells became more common after acoustic trauma. The timing of the onset of delayed responses suggests that these could occur via G-protein coupled receptor pathways and/or post-translational modifications, although the specific mechanism is unknown (Hoare et al, 2020;Gold, 1999). Increased somatosensory pain can occur within the same time frame (2-5 min) as the delayed cochlear responses to acute tissue damage after acoustic trauma.…”