Elusive equilibrium: the challenge of interpreting receptor pharmacology using calcium assays (original) (raw)
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FEBS Letters, 1996
nic m3 receptor plays a dominant role in the regulation of Abstract The m2 and m3 muscarinic acetylcholine receptors Ca 2+ influx in CHO cells. were expressed in CHO cells and were shown to couple to the release of calcium from intracellular stores. The m3 receptor, but not the m2 receptor, also coupled to calcium influx. Chimeric m21 2. Materials and methods m3 receptors were used to determine the structural domain of the m3 receptor linked to the regulation of calcium influx. It was 2.1. Chimeric eDNA construct preparation and cell culture found that the third intracellular loop of m3 receptor plays a
British Journal of Pharmacology, 1995
1 Phosphoinositidase C-linked m3-muscarinic receptors expressed in Chinese hamster ovary cells (CHO-m3 cells) are phosphorylated on serine following agonist stimulation. 2 m3-Muscarinic receptor phosphorylation is concentration-dependent requiring a carbachol concentration of 13.2 /M for half maximal stimulation. 3 The phosphorylation concentration-response curve lies to the left of the curve for carbachol binding to muscarinic receptors (KD = 100 gIM) in membranes from CHO-m3 cells. In contrast, receptor phosphorylation closely correlates with receptor-mediated phosphoinositidase C activation (EC50 for inositol 1,4,5 trisphosphate accumulation during the peak and plateau phases were 7.14 ,M and 5.92 ,UM respectively) but not with rapid agonist-mediated calcium elevation (EC5o = 0.32 gM) measured in fura-2-AM loaded cells. 4 These data suggest a dissociation of receptor phosphorylation from agonist occupation. Such an apparent 'receptor reserve' for m3-muscarinic receptor phosphorylation may be indicative of a mechanism that is dependent on a small amplification of the receptor signal, though probably dissociated from the calcium signal.
Journal of Pharmacological and Toxicological Methods, 2014
Introduction: The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its capacity to activate downstream signaling events. In this report, we describe a reverse-engineering strategy for estimating these microscopic constants. Methods: Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein-coupled receptor under conditions of allosteric modulation and reduced receptor expression or partial receptor inactivation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligandsincluding inverse agonistsand the efficacy of the unoccupied receptor (i.e., constitutive activity). Results and discussion: We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor-signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe-dependent allosteric interactions and the selectivity of agonists for different signaling pathways. Knowing the isomerization constant and sensitivity constant of a signaling pathway in a given cell line or tissue preparation enables future investigators to estimate the affinity constants of agonists for receptor states simply through analysis of their concentration-response curves. Our approach also provides a means of validating in silico estimates of ligand affinity for crystal structures of active and inactive states of the receptor. j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j p h a r m t o x ð Þ KE−obs (Domain, K q − obs N 0; units, inverse concentration, e.g., M −1 )
Biased agonism of the calcium-sensing receptor
After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby " biasing " the signaling, it has become obvious that the original model of GPCRs existing in either an " on " or " off " conformation is too simple. The current explanation for this biased agonism is that GPCRs can adopt multiple active confor-mations stabilized by different molecules, and that each conformation affects intracellular signaling in a different way. In the present study we sought to investigate biased agonism of the calcium-sensing receptor (CaSR), by looking at 12 well-known orthosteric CaSR agonists in 3 different CaSR signaling pathways: G q/11 protein, G i/o protein, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Here we show that apart from G q/11 and G i/o signaling, ERK1/2 is activated through recruitment of-arrestins. Next, by measuring activity of all three signaling pathways we found that barium, spermine, neomycin, and tobramycin act as biased agonist in terms of efficacy and/or potency. Finally, polyamines and amino-glycosides in general were biased in their potencies toward ERK1/2 signaling. In conclusion, the results of this study indicate that several active conformations of CaSR, stabilized by different molecules, exist, which affect intracellular signaling distinctly.
The FASEB Journal, 2008
We describe a simple method for calculating the pharmacological activity of an agonist (A) relative to a standard agonist (S) using only the concentration-response curves of the two agonists. In most situations, we show that the product of the ratios of maximal responses (E max Ϫ A /E max Ϫ S) and potencies (EC 50 Ϫ S /EC 50 Ϫ A) is equivalent to the product of the affinity and intrinsic efficacy of A expressed relative to that of S. We refer to this term as the IRA value of A. In a cooperative system where the concentrationresponse curve of the standard agonist is steep and that of the test agonist is flatter with a lower maximal response, the simple calculation of IRA described above underestimates agonist activity; however, we also describe a means of correcting the IRA in this situation. We have validated our analysis with modeling techniques and have shown experimentally that the IRA values of muscarinic agonists for stimulating contractions in the guinea pig ileum (M 3 response) are in excellent agreement with those measured in the phosphoinositide assay on Chinese hamster ovary cells expressing the M 3 muscarinic receptor.
Molecular pharmacology, 2000
The quantitative comparison of the relative potency of agonists is a standard method of receptor and agonist classification. If agonist potency ratios do not correspond in two given tissues, this is used as presumptive data to conclude that the receptors in those two tissues are different. This article presents data to show that a single receptor can demonstrate varying agonist potency ratios in different host cells. These data are described in terms of the production of more than one agonist-selective receptor active state and the interaction of these different active states with multiple G proteins in the membrane to produce cellular response. Stable host human embryonic kidney 293 cells with enhanced quantities of the respective Galpha-protein were created. Wild-type and Galpha-subunit enriched cells were then transiently transfected with human calcitonin receptor type 2 (hCTR2). Binding did not detect differences in the G protein-enriched cells versus wild-type cells. In contras...
Journal of Pharmacology and Experimental Therapeutics, 2007
We developed novel methods for analyzing the concentrationresponse curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA i ), is most applicable for the analysis of responses at G protein-coupled receptors. RA i is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M 2 receptor, including stimulation of phosphoinositide hydrolysis through G ␣15 in HEK 293T cells, inhibition of cAMP accumulation through G i in Chinese hamster 1 Here and throughout the article, we use the term "observed intrinsic efficacy" to refer to Furchgott's definition of "intrinsic efficacy" , which denotes the amount of activated receptors. As described previously , it is useful to discriminate between the latter definition of observed intrinsic efficacy and the ratio of agonist affinity constants for ground and active conformations of the receptor (intrinsic efficacy).
2011
We describe a modification of receptor theory that enables the estimation of relative affinity constants for the inactive state of a G protein-coupled receptor. Our approach includes the traditional parameters of observed affinity (K obs ) and efficacy (fraction of ligand-receptor complex in the active state, ) and introduces the concept of the fraction of the ligand-receptor complex in the inactive state (intrinsic inactivity, i ). The relationship between receptor activation and the ligand concentration is known as the stimulus, and the operational model expresses the response as a logistic function of the stimulus. The latter function includes K obs and the parameter , which is proportional to . We introduce the parameter i , which is proportional to i . We have previously shown that the product, K obs , of one agonist, expressed relative to that of another (intrinsic relative activity, RA i ), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, K obs i , of one agonist, expressed relative to that of another (intrinsic relative inactivity, RI i ), is a relative measure of the affinity constant for the inactive state of the receptor. We use computer simulation techniques to verify our analysis and apply our method to the analysis of published data on agonist activity at the M 3 muscarinic receptor. Our method should have widespread application in the analysis of agonist bias in drug discovery programs and in the estimation of a more fundamental relative measure of efficacy (RA i /RI i ).
Journal of Pharmacology and Experimental Therapeutics, 2011
We describe a modification of receptor theory that enables the estimation of relative affinity constants for the inactive state of a G protein-coupled receptor. Our approach includes the traditional parameters of observed affinity (K obs ) and efficacy (fraction of ligand-receptor complex in the active state, ) and introduces the concept of the fraction of the ligand-receptor complex in the inactive state (intrinsic inactivity, i ). The relationship between receptor activation and the ligand concentration is known as the stimulus, and the operational model expresses the response as a logistic function of the stimulus. The latter function includes K obs and the parameter , which is proportional to . We introduce the parameter i , which is proportional to i . We have previously shown that the product, K obs , of one agonist, expressed relative to that of another (intrinsic relative activity, RA i ), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, K obs i , of one agonist, expressed relative to that of another (intrinsic relative inactivity, RI i ), is a relative measure of the affinity constant for the inactive state of the receptor. We use computer simulation techniques to verify our analysis and apply our method to the analysis of published data on agonist activity at the M 3 muscarinic receptor. Our method should have widespread application in the analysis of agonist bias in drug discovery programs and in the estimation of a more fundamental relative measure of efficacy (RA i /RI i ).