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![Recovery of the TRH response in 301 cells. Changes in IP 3 , [Ca 2 ] i , and the intracellular Ca 2 pool are shown schematically. The dotted line depicts the initial response to TRH. Arrows denote responses to single applications of TRH given at 10, 20, 30, or 40 min after washing out the TRH. The experiment could not be extended unless cells were loaded with Fura-2 during the recovery period. When this was done, Ca 2 pools were found to refill in 1 h.](publication/13876794/figure/fig6/AS:1022893215469575@1620888155362/Recovery-of-the-TRH-response-in-301-cells-Changes-in-IP-3-Ca-2-i-and-the.png)
Recovery of the TRH response in 301 cells. Changes in IP 3 , [Ca 2 ] i , and the intracellular Ca 2 pool are shown schematically. The dotted line depicts the initial response to TRH. Arrows denote responses to single applications of TRH given at 10, 20, 30, or 40 min after washing out the TRH. The experiment could not be extended unless cells were loaded with Fura-2 during the recovery period. When this was done, Ca 2 pools were found to refill in 1 h.
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![FIG. 1. Typical single cell [Ca 2 ] i responses of 301 cells to TRH....](publication/13876794/figure/fig1/AS:1022893215477760@1620888155279/Typical-single-cell-Ca-2-i-responses-of-301-cells-to-TRH-Fura-2-loaded-cells-were_Q320.jpg)
![FIG. 3. Concentration dependence of TRH-mediated increases in [Ca 2 ] i...](publication/13876794/figure/fig3/AS:1022893215465473@1620888155312/Concentration-dependence-of-TRH-mediated-increases-in-Ca-2-i-and-IP-3-in-301-cells_Q320.jpg)
Desensitization and recovery of the inositol 1,4,5-trisphosphate (IP3) and intracellular free calcium concentration ([Ca2+]i) responses to thyrotropin-releasing hormone (TRH) were measured in HEK293 cells stably expressing the G protein-coupled TRH
receptor. TRH caused a large, rapid, and transient increase in IP3 and a biphasic increase in [Ca2+]i...
Citations
... An unusual regulation mechanism is involved in Ca 2+ signaling via intracellular stores. The amount of cytoplasmic Ca 2+ that can be obtained is limited by the amount of Ca 2+ in the intracellular stores 66,67 . Depletion of Ca 2+ in the store as it is being released into the cytoplasm limits the amount and rate of cytoplasmic Ca 2+ release. ...
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.
... An unusual regulation mechanism is involved in Ca 2+ signaling via intracellular stores. The amount of cytoplasmic Ca 2+ that can be obtained is limited by the amount of Ca 2+ in the intracellular stores 57,58 . Depletion of Ca 2+ in the store as it is being released into the cytoplasm limits the amount and rate of cytoplasmic Ca 2+ release. ...
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 (kTau), 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.
... Response generation is the increase of cytoplasmic Ca 2+ concentration resulting from opening of the IP3-gated Ca 2+ channel in the ER membrane (Berridge, 1993;Berridge et al., 2003). Precursor depletion is the decrease of Ca 2+ in the ER resulting from Ca 2+ release into the cytoplasm (Hofer et al., 1998;Miyawaki et al., 1997;Yu and Hinkle, 1997; . CC-BY 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
Pharmacological responses are modulated over time by regulation of signaling mechanisms. The canonical short-term regulation mechanisms are receptor desensitization and degradation of the response. Here for the first time a pharmacological model for measuring drug parameters is developed that incorporates short-term mechanisms of regulation of signaling. The model is formulated in a manner that enables measurement of drug parameters using familiar curve fitting methods. The efficacy parameter is k τ , which is simply the initial rate of signaling before it becomes limited by regulation mechanisms. The regulation parameters are rate constants, k DES for receptor desensitization and k D for response degradation. Efficacy and regulation are separate parameters, meaning these properties can be optimized independently of one another in drug discovery. The parameters can be applied to translate in vitro findings to in vivo efficacy in terms of the magnitude and duration of drug effect. When the time course data conform to certain shapes, for example the association exponential curve, a mechanism-agnostic approach can be applied to estimate agonist efficacy, without the need to know the underlying regulatory mechanisms. The model was verified by comparison with historical data and by fitting these data to estimate the model parameters. This new model for quantifying drug activity can be broadly applied to the short-term cell signaling assays used routinely in drug discovery and to aid their translation to in vivo efficacy, facilitating the development of new therapeutics.
Highlights
Regulation of signaling impacts measurement of drug effect
Receptor desensitization is incorporated here into a kinetic model of signaling
Drug effect and signaling regulation can now be measured independently
The analysis framework is designed for signaling assays used in drug discovery
These new analysis capabilities will aid development of new therapeutics
... The extent of desensitization depends on the method used to measure it and the cell type under study (Falck-Pedersen et al., 1994). Decoupling the receptor from G proteins contributes to the transient nature of the IP3 elevation, which is more sustained in cells expressing a TRH receptor that lacks most of the cytoplasmic tail and is thereby spared from usual desensitization processes Jones and Hinkle, 2005), as shown in Figure 3. Changes further downstream in the signal pathway can also decrease TRH responses by mechanisms including protein kinase C-mediated inhibition of phospholipase Cβ and slow refilling of intracellular calcium stores (Yu and Hinkle, 1997). TRH receptors were the first GPCRs shown to undergo what is now termed homologous downregulation (Hinkle and Tashjian, 1975;Gershengorn, 1978). ...
The pituitary receptor for thyrotropin-releasing hormone (TRH) is a calcium-mobilizing G protein-coupled receptor (GPCR) that signals through Gq/11, elevating calcium, and activating protein kinase C. TRH receptor signaling is quickly desensitized as a consequence of receptor phosphorylation, arrestin binding, and internalization. Following activation, TRH receptors are phosphorylated at multiple Ser/Thr residues in the cytoplasmic tail. Phosphorylation catalyzed by GPCR kinase 2 (GRK2) takes place rapidly, reaching a maximum within seconds. Arrestins bind to two phosphorylated regions, but only arrestin bound to the proximal region causes desensitization and internalization. Phosphorylation at Thr365 is critical for these responses. TRH receptors internalize in clathrin-coated vesicles with bound arrestin. Following endocytosis, vesicles containing phosphorylated TRH receptors soon merge with rab5-positive vesicles. Over approximately 20 min these form larger endosomes rich in rab4 and rab5, early sorting endosomes. After TRH is removed from the medium, dephosphorylated receptors start to accumulate in rab4-positive, rab5-negative recycling endosomes. The mechanisms responsible for sorting dephosphorylated receptors to recycling endosomes are unknown. TRH receptors from internal pools help repopulate the plasma membrane. Dephosphorylation of TRH receptors begins when TRH is removed from the medium regardless of receptor localization, although dephosphorylation is fastest when the receptor is on the plasma membrane. Protein phosphatase 1 is involved in dephosphorylation but the details of how the enzyme is targeted to the receptor remain obscure. It is likely that future studies will identify biased ligands for the TRH receptor, novel arrestin-dependent signaling pathways, mechanisms responsible for targeting kinases and phosphatases to the receptor, and principles governing receptor trafficking.
... These compounds can be readily detected in the agonist mode portion of the screen. However, the agonist response can result in receptor internalization/desensitization, depletion of the detection reagent, and/or emptying of the Ca 2+ stores, which in the continued presence of the compound do not re-fill [34]. This can result in a desensitization of the Ca 2+ response to further challenges with agonists or PAMs. ...
Once considered a pharmacological curiosity, allosteric modulation of seven transmembrane domain G-protein-coupled receptors (GPCRs) has emerged as a potentially powerful means to affect receptor function for therapeutic purposes. Allosteric modulators, which interact with binding sites topologically distinct from the orthosteric ligand binding sites, can potentially provide improved selectivity and safety, along with maintenance of spatial and temporal aspects of GPCR signaling. Accordingly, drug discovery efforts for GPCRs have increasingly focused on the identification of allosteric modulators. This review is devoted to an examination of the strategies, challenges, and opportunities for high-throughput screening for allosteric modulators of GPCRs, with particular focus on the identification of positive allosteric modulators.
... We recorded Ca 2+ responses to a 1-min challenge of TRH (100 nM). Consecutive TRH applications were given 30 min apart to allow ER stores to replenish between applications (35). Each challenge of TRH was applied in the absence of extracellular Ca 2+ (removed 5 min before and added back 5 min after TRH treatment), to prevent Ca 2+ influx into the cell during the stimula-tion. ...
Cell responses are commonly heterogeneous, even within a subpopulation. In the present study, we investigate the source of heterogeneity in the Ca(2+) response of anterior pituitary lactotrophs to a Ca(2+) mobilisation agonist, thyrotrophin-releasing hormone. This response is characterised by a sharp increase of cytosolic Ca(2+) concentration as a result of mobilisation of Ca(2+) from intracellular stores, followed by a decrease to an elevated plateau level that results from Ca(2+) influx. We focus on heterogeneity of the evoked Ca(2+) spike under extracellular Ca(2+) free conditions. We introduce a method that uses the information provided by a mathematical model to characterise the source of heterogeneity. This method compares scatter plots of features of the Ca(2+) response obtained experimentally with those made from the mathematical model. The model scatter plots reflect random variation of parameters over different ranges, and matching the experimental and model scatter plots allows us to predict which parameters are most variable. We find that a large degree of variation in Ca(2+) efflux is a likely key contributor to the heterogeneity of Ca(2+) responses to thyrotrophin-releasing hormone in lactotrophs. This technique is applicable to any situation in which the heterogeneous biological response is described by a mathematical model.
... TRH is a tripeptide that functions as a hormone regulating TSH and prolactin secretion. TRH receptors signal through G q and G 11 , leading to the production of inositol (1,4,5) triphosphate and the release of intracellular calcium. After TRH binds, the TRH receptor is rapidly desensitized (1). ...
... TRH receptors signal through G q and G 11 , leading to the production of inositol (1,4,5) triphosphate and the release of intracellular calcium. After TRH binds, the TRH receptor is rapidly desensitized (1). Desensitization of GPCRs is initiated when receptors are phosphorylated by G protein-coupled receptor kinases (GRKs) or second messenger-activated kinases. ...
The G protein-coupled thyrotropin (TSH)-releasing hormone (TRH) receptor forms homodimers. Regulated receptor dimerization increases TRH-induced receptor endocytosis. These studies test whether dimerization increases receptor phosphorylation, which could potentiate internalization. Phosphorylation at residues 355–365, which is critical for internalization, was measured with a highly selective phospho-site-specific antibody. Two strategies were used to drive receptor dimerization. Dimerization of a TRH receptor-FK506-binding protein (FKBP) fusion protein was stimulated by a dimeric FKBP ligand. The chemical dimerizer caused a large increase in TRH-dependent phosphorylation within 1 min, whereas a monomeric FKBP ligand had no effect. The dimerizer did not alter phoshorylation of receptors lacking the FKBP domain. Dimerization of receptors containing an N-terminal HA epitope also was induced with anti-HA antibody. Anti-HA IgG strongly increased TRH-induced phosphorylation, whereas monomeric Fab fragments had no effect. Anti-HA antibody did not alter phosphorylation in receptors lacking an HA tag. Furthermore, two phosphorylation-defective TRH receptors functionally complemented one another and permitted phosphorylation. Receptors with a D71A mutation in the second transmembrane domain do not signal, whereas receptors with four Ala mutations in the 355–365 region signal normally but lack phosphorylation sites. When D71A- and 4Ala-TRH receptors were expressed alone, neither underwent TRH-dependent phosphorylation. When they were expressed together, D71A receptor was phosphorylated by G protein-coupled receptor kinases in response to TRH. These results suggest that the TRH receptor is phosphorylated preferentially when it is in dimers or when preexisting receptor dimers are driven into microaggregates. Increased receptor phosphorylation may amplify desensitization.
• G protein
• desensitization
• G protein-coupled receptor
• G protein-coupled receptor kinase
• PKC
... To determine the mechanism by which the calcium signal desensitized, we tested potential mediators of this effect. First, because depletion of intracellular calcium stores by prolonged agonist treatment is a mediator of desensitization of some G q/11 protein coupled receptors (Yu and Hinkle, 1997), we sought to determine whether the amount of intracellular calcium remaining was a limiting factor contributing to lowered calcium responses by quantifying intracellular calcium release after agonist pretreatment. The sarcoplasmic endoplasmic reticulum Ca 2ϩ -ATPase inhibitor thapsigargin (1 M) was added in the presence of the extracellular calcium chelator EGTA to quantify intracellular calcium levels in sarcoplasmic endoplasmic reticulum Ca 2ϩ -ATPase-controlled stores after treatment with various agonists and compared with control levels. ...
When dopamine D1 and D2 receptors were coactivated in D1-D2 receptor hetero-oligomeric complexes, a novel phospholipase C-mediated calcium signal was generated. In this report, desensitization of this Gq/11-mediated calcium signal was demonstrated by pretreatment with dopamine or with the D1-selective agonist (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297) or the D2-selective agonist quinpirole. Desensitization of the calcium signal mediated by D1-D2 receptor hetero-oligomers was initiated by agonist occupancy of either receptor subtype even though the signal was generated only by occupancy of both receptors. The efficacy, potency, and rate of calcium signal desensitization by agonist occupancy of the D1 receptor (t1/2, approximately 1 min) was far greater than by the D2 receptor (t1/2, approximately 10 min). Desensitization of the calcium signal was not mediated by depletion of calcium stores or internalization of the hetero-oligomer and was not decreased by inhibiting second messenger-activated kinases. The involvement of G protein-coupled receptor kinases 2 or 3, but not 5 or 6, in the desensitization of the calcium signal was shown, occurring through a phosphorylation independent mechanism. Inhibition of Gi protein function associated with D2 receptors increased D1 receptor-mediated desensitization of the calcium signal, suggesting that cross-talk between the signals mediated by the activation of different G proteins controlled the efficacy of calcium signal desensitization. Together, these results demonstrate the desensitization of a signal mediated only by hetero-oligomerization of two G protein-coupled receptors that was initiated by agonist occupancy of either receptor within the hetero-oligomer, albeit with differences in desensitization profiles observed.
... In pituitary cells expressing TRH receptors, continuous exposure to TRH causes an increase in IP 3 concentration that peaks within 10 s, but then falls within 1 min to remain at approximately twice the basal level for at least 10 min (4). Desensitization of the TRH response is rapid and controlled upstream of phospholipase C activity (4,34), most likely because the receptor is uncoupled from G protein as a consequence of phosphorylation and -arrestin binding. In the present experiments, we anticipated that IP 3 production might begin at similar rates in cells with or without -arrestin, and then slow down in cells expressing -arres-tin. ...
The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to β-arrestin after agonist
exposure. To define the importance of receptor phosphorylation and β-arrestin binding in desensitization, and to determine
whether β-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors
in fibroblasts from mice lacking β-arrestin-1 and/or β-arrestin-2. Apparent affinity for [3H]MeTRH was increased 8-fold in cells expressing β-arrestins, including a β-arrestin mutant that did not permit receptor internalization.
TRH caused extensive receptor endocytosis in the presence of β-arrestins, but receptors remained primarily on the plasma membrane
without β-arrestin. β-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous
β-arrestins reduced TRH-stimulated inositol phosphate production by 48% (β-arrestin-1), 71% (β-arrestin-2), and 84% (β-arrestins-1
and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected
by β-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical
with or without β-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose
did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Gαq and Gα11 or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression
of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation
caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation
can take place on the plasma membrane and that β-arrestin binding is critical for desensitization and internalization.
... Wortmannin does not completely inhibit the early Ins(1,4,5)P 3 response triggered by a diverse number of receptors [17,18] ; therefore, wortmannin-induced inhibition of Ca 2; release triggered by histamine also explains the lack of the initial Ins(1,4,5)P 3 response evoked by histamine as this response depends on Ca 2; release from internal stores. Furthermore, despite the evidence of Ins(1,4,5)P 3 being a long-range second messenger [44], our data support the notion that Ins(1,4,5)P 3 works locally [45,46], as the Ins(1,4,5)P 3 involved in releasing Ca 2; by saturating concentrations of histamine was not detected. One possibility is that histamine H 1 receptors do not need to increase Ins(1,4,5)P 3 levels, but activate PLC to remove PIP 2 from the IP 3 Rs to disinhibit these channels; a possibility derived from a recently proposed model for the activation of IP 3 R [47]. ...
We have studied the Ca(2+)-dependence and wortmannin-sensitivity of the initial inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) response induced by activation of either histamine or muscarinic receptors in smooth muscle from guinea pig urinary bladder. Activation of H(1) receptors with histamine (100 microM) produced a significant elevation in Ins(1,4,5)P(3) levels with only 5s stimulation and in the presence of external Ca(2+). However, this response was abolished fully by either the prolonged absence of external Ca(2+) or the depletion of internal Ca(2+) stores with thapsigargin (100nM) or ryanodine (10 microM). In contrast, the same conditions only slightly reduced the initial Ins(1,4,5)P(3) response induced by carbachol. The prolonged incubation of smooth muscle in 10 microM wortmannin to inhibit type III PI 4-kinase abolished both the early histamine-evoked Ins(1,4,5)P(3) and Ca(2+) responses. Conversely, wortmannin did not alter Ca(2+) release induced by carbachol, despite a partial reduction of its Ins(1,4,5)P(3) response. Collectively, these data indicate that the detectable histamine-induced increase in Ins(1,4,5)P(3) is more the consequence of Ca(2+) release from internal stores than a direct activation of phospholipase C by H(1) receptors. In addition, the effect of wortmannin implies the existence of a Ca(2+)-dependent amplification loop for the histamine-induced Ins(1,4,5)P(3) response in smooth muscle.
