Why classical receptor theory, which ignores allostery, can effectively measure the strength of an allosteric effect as agonist's efficacy.

BACKGROUND AND PURPOSE: The classical theory of receptor action has been used for decades as a powerful tool to estimate molecular determinants of ligand-induced receptor activation (i.e., affinity and efficacy) from experimentally observable biological responses. However, it is also a well-recognized fact that the receptor-binding and activation mechanisms, and the parameters thereof, described in the classical theory contradict with the modern view of receptor activation based on allosteric principles. EXPERIMENTAL APPROACH: We used mathematical analysis, along with some numerical simulations, to answer the key question as to what extent the classical theory is compatible-if at all-with the modern understanding of receptor activation. KEY RESULTS: Here, we showed conclusively that (1) receptor activation equations based on allosteric principles contain the logic of the classical theory in disguise, and therefore, (2) estimates of "intrinsic efficacy" (ε) obtained by means of classical techniques (i.e., null methods or fitting the operational model to concentration-response data) are equivalent to the allosteric coupling factors that represent the molecular efficacy of ligands. CONCLUSION AND IMPLICATIONS: Thus, we conclude that despite the justified criticisms it has received so far, the classical theory may continue to be useful in estimating ligand efficacy from experimental data, if used properly. Here, we also provide rigorous criteria for the proper use of the theory. These findings not only have implications for ligand classification but also resolve some long lasting discussions in the field of bias agonism in GPCR, which requires reasonable estimates of relative ligand efficacies at different signalling pathways.

Community guidelines for GPCR ligand bias: IUPHAR review 32.

GPCRs modulate a plethora of physiological processes and mediate the effects of one-third of FDA-approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular transducers. This 'biased signalling' paradigm requires that we now characterize physiological signalling not just by receptors but by ligand-receptor pairs. Ligands eliciting biased signalling may constitute better drugs with higher efficacy and fewer adverse effects. However, ligand bias is very complex, making reproducibility and description challenging. Here, we provide guidelines and terminology for any scientists to design and report ligand bias experiments. The guidelines will aid consistency and clarity, as the basic receptor research and drug discovery communities continue to advance our understanding and exploitation of ligand bias. Scientific insight, biosensors, and analytical methods are still evolving and should benefit from and contribute to the implementation of the guidelines, together improving translation from in vitro to disease-relevant in vivo models.

Biased agonism at G protein-coupled receptors.

G protein-coupled receptors (GPCRs) represent the largest family of approved therapeutic targets. Ligands stimulating these receptors specifically activate multiple signalling pathways that induce not only the desired therapeutic response, but sometimes untolerated side effects that limit their clinical use. The diversity in signalling induced by each ligand could be considered a viable path for improving this situation. Biased agonism, which offers the promise of identifying pathway-selective drugs has been proposed as a means to exploit this opportunity. However, identifying biased agonists is not an easy process and quantifying ligand bias for a given signalling pathway requires careful consideration and control of several confounding factors. To date, the molecular mechanisms of biased signalling remain unclear and known theories that constitute our understanding of the mechanisms underlying therapeutic and side effects are still being challenged, making the strategy of selecting promising potential drugs more difficult. This special issue summarizes the latest advances in the discovery and optimization of biased ligands for different GPCRs. It also focuses on identifying novel insights into the field of biased agonism, while at the same time, highlighting the conceptual and experimental limitations of that concept for drug discovery. This aims to broaden our understanding of the signalling induced by the various identified biased agonists and provide perspectives that could straighten our path towards the development of more effective and tolerable therapeutics.

Conceptual and experimental issues in biased agonism.

In this review, we discuss the theoretical and experimental foundations for assessing agonism in the context of signalling bias in GPCRs. We show that the formulation of efficacy in classical receptor theory and the definition of ligand-induced allosteric effect in chemical thermodynamics are coincident measures of agonism, only if we recognize that the classical model cannot be considered as a mechanistic description of the physicochemical events underlying ligand-receptor signalling. It represents instead a mathematical tool, fortuitously capable of extracting efficacy information from concentration-dependent functional data, where both ligand-dependent and ligand-independent information are present. We also assert that dissecting efficacy from affinity, as originally advocated in classical theory, is imperative for understanding the molecular property underlying agonism, and the biased agonism that leads to preferential formation of diverse GPCR-transducer complexes. Finally, we argue that beyond the assumed translational value of functional selectivity (i.e. signalling bias), the identification of ligands with true bias of efficacy is of fundamental importance for unravelling the conformational space that determines the complex functional chemistry of GPCRs.

Vasopressin receptor 2 mutations in the nephrogenic syndrome of inappropriate antidiuresis show different mechanisms of constitutive activation for G protein coupled receptors.

Vasopressin receptor 2 (V2R) mutations causing the nephrogenic syndrome of inappropriate antidiuresis (NSIAD) can generate two constitutively active receptor phenotypes. One type results from residue substitutions in several V2R domains and is sensitive to vaptan inverse agonists. The other is only caused by Arg 137 replacements and is vaptan resistant. We compared constitutive and agonist-driven interactions of the vaptan-sensitive F229V and vaptan-resistant R137C/L V2R mutations with ß-arrestin 1, ß-arrestin 2, and Gαs, using null fibroblasts reconstituted with individual versions of the ablated transduction protein genes. F229V displayed very high level of constitutive activation for Gs but not for ß-arrestins, and enhanced or normal responsiveness to agonists and inverse agonists. In contrast, R137C/L mutants exhibited maximal levels of constitutive activation for ßarrestin 2 and Gs, minimal levels for ß-arrestin 1, but a sharp decline of ligands sensitivity at all transducer interactions. The enhanced constitutive activity and reduced ligand sensitivity of R137 mutants on cAMP signaling persisted in cells lacking ß-arrestins, indicating that these are intrinsic molecular properties of the mutations, not the consequence of altered receptor trafficking. The results suggest that the two groups of NSIAD mutations represent two distinct molecular mechanisms of constitutive activation in GPCRs.

Systematic errors in detecting biased agonism: Analysis of current methods and development of a new model-free approach.

Discovering biased agonists requires a method that can reliably distinguish the bias in signalling due to unbalanced activation of diverse transduction proteins from that of differential amplification inherent to the system being studied, which invariably results from the non-linear nature of biological signalling networks and their measurement. We have systematically compared the performance of seven methods of bias diagnostics, all of which are based on the analysis of concentration-response curves of ligands according to classical receptor theory. We computed bias factors for a number of ß-adrenergic agonists by comparing BRET assays of receptor-transducer interactions with Gs, Gi and arrestin. Using the same ligands, we also compared responses at signalling steps originated from the same receptor-transducer interaction, among which no biased efficacy is theoretically possible. In either case, we found a high level of false positive results and a general lack of correlation among methods. Altogether this analysis shows that all tested methods, including some of the most widely used in the literature, fail to distinguish true ligand bias from "system bias" with confidence. We also propose two novel semi quantitative methods of bias diagnostics that appear to be more robust and reliable than currently available strategies.

What is biased efficacy? Defining the relationship between intrinsic efficacy and free energy coupling.

A G protein-coupled receptor (GPCR) is only biologically active when associated with a transduction protein, but it can also switch function by interacting with different types of transduction proteins. Biased agonism arises when the ligand induces the receptor to engage distinct transduction proteins with different efficacies. We briefly review the concept of ligand efficacy, from the classical empirical idea to the current mechanistic views of allosteric regulation in proteins. A combination of these theoretically distinct ideas and methodologies allows us to distinguish true ligand bias from divergences of signalling caused by the system. We also demonstrate a rigorous mathematical connection between the intrinsic efficacy of classical receptor theory and the energetic effect that makes a ligand capable of stabilizing receptor-transducer association in the ternary complex model. This relationship unifies different definitions of efficacy and provides a rational basis for quantifying biased agonism.

Ligands raise the constraint that limits constitutive activation in G protein-coupled opioid receptors.

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and µ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

Where have all the active receptor states gone?

Defining G protein-coupled receptor ligand efficacy and biased agonism in precise chemical terms is one challenge posed by the current structural data that exists for this receptor family. Concepts classically used for understanding enzymes and other nonreceptor proteins may lead us in the right direction.

Cell contact-dependent functional selectivity of ß2-adrenergic receptor ligands in stimulating cAMP accumulation and extracellular signal-regulated kinase phosphorylation.

Activation of ß(2)-adrenegic receptor (ß(2)-AR) leads to an increase in intracellular cAMP and activation of ERK. These two signals are activated by the interaction of the receptor with different transducer partners. We showed that the intrinsic activities of ß(2)-AR ligands for stimulating cAMP production and ERK phosphorylation responses in HEK-293 cells were not correlated. The lack of correlation resulted mainly from the discrepancy between the intrinsic activities of two groups of ligands for these two responses: The first group consisted of clenbuterol, cimaterol, procaterol, and terbutaline which acted as full agonists for cAMP production but displayed very weak effect on ERK phosphorylation. The second group comprised adrenaline and noradrenaline which displayed higher intrinsic activity for the ERK phosphorylation than for the cAMP response. Thus, both groups behaved as functionally selective ligands. The functional selectivity of the first group was observable only in adherent cells when confluence was approximately 100%. When cell-cell contact was minimized either by decreasing the density of the adherent cells or by bringing the cells into suspension, the first group of ligands gained the ability to stimulate ERK phosphorylation without a change in their effect on cAMP production. In contrast, selectivity of the second group was independent of the adherence state of the cells. Our results show that the inherent "bias" of ligands in coupling a G protein-coupled receptor to different transducers may not always be revealed as functional selectivity when there is a "cross-talk" between the signaling pathways activated by the same receptor.

Long and short distance movements of ß(2)-adrenoceptor in cell membrane assessed by photoconvertible fluorescent protein dendra2-ß(2)-adrenoceptor fusion.

Local movements of receptors in the plasma membrane have been extensively studied, as it is generally believed that the dynamics of membrane distribution of receptors regulate their functions. However, the properties of large-scale (>5µm) receptor movements in the membrane are relatively obscure. In the present study, we addressed the question as to whether the large-scale movement of receptor in the plasma membrane at the whole cell level can be explained quantitatively by its local diffusive properties. We used HEK 293 cells transfected with human ß2-adrenoceptor fused to photoconvertible fluorescent protein dendra2 as a model system; and found that 1) functional integrity of the dendra2-tagged receptor remains apparently intact; 2) in a mesoscopic scale (~4µm), ~90% of the receptors are mobile on average, and receptor influx to, and out-flux from a membrane area can be symmetrically explained by a diffusion-like process with an effective diffusion coefficient of ~0.1µm(2)/s; 3) these mobility parameters are not affected by the activity state of the receptor (assessed by using constitutively active receptor mutants); 4) in the macroscopic scale (4-40µm), although a slowly diffusing fraction of receptors (with D<0.01µm(2)/s) is identifiable in some cases, the movement of the predominant fraction is perfectly explained by the same effective diffusion process observed in the mesoscopic scale, suggesting that the large scale structure of the cell membrane as felt by the receptor is apparently homogeneous in terms of its mesoscopic properties. We also showed that intracellular compartments and plasma membrane are kinetically connected even at steady-state.

beta2-Adrenoceptor, Gs and adenylate cyclase coupling in purified detergent-resistant, low density membrane fractions.

Membrane rafts and caveolae are specialized microdomains of the cell membrane that form physical platforms for compartmentalization of signalling molecules. Here, we intended to gain insight into the consequences of caveolar localization in G protein-coupled receptor function. We analysed beta(2)-adrenoceptor signalling in purified CRLDF (caveolin-rich low density fractions) of beta(2)-adrenoceptor-overexpressing HEK-293 cells. beta(2)-adrenoceptor and Gs immunoreactivities and forskolin-stimulated adenylate cyclase activity were all detected in CRLDF obtained by the conventional raft purification method that uses Triton X-100 solubilization. However, Triton X-100 caused a complete loss of the functional coupling between beta(2)-adrenoceptor, Gs and adenylate cyclase. Therefore, we developed an optimized purification method based on n-octyl-beta-d-glucopyranoside solubilization, where the functional properties of beta(2)-adrenoceptor, Gs and adenylate cyclase were preserved in the CRLDF. Using this method, we showed that isoproterenol-stimulated adenylate cyclase activity was similar in CRLDF and bulk membrane preparations of HEK-293 cells that overexpress beta(2)-adrenoceptor or beta(2)-adrenoceptor-Gs fusion. Accordingly, treatment of cells with methyl-beta-cyclodextrin, a caveola-disrupting agent, did not affect beta(2)-adrenoceptor-induced cAMP response. Likewise, these responses were insensitive to caveolin 1 and 2 overexpression. On the other hand, methyl-beta-cyclodextrin treatment did decrease beta(2)-adrenoceptor-induced ERK phosphorylation. However, the latter effect of methyl-beta-cyclodextrin could be attributed to a non-specific effect rather than its ability to disrupt membrane microdomains. We showed that localization in the raft microdomains did not affect the signalling efficiency of beta(2)-adrenoceptor-Gs-adenylate cyclase pathway, and that methyl-beta-cyclodextrin may inhibit signalling by directly affecting the signalling system independently of its caveola-disrupting property.

Allosteric coupling and conformational fluctuations in proteins.

Proteins in their native folded states can possess multiple energy minima and they can show constant conformational fluctuations at physiological temperatures. In this article, we discuss the quantitative relationship between ligand-induced perturbation of such fluctuations, modeled as probability distributions of conformational substates, and allosteric coupling of ligand binding to different sites, as defined by linkage thermodynamics. We show that allosteric coupling between two binding events on the same protein is an inevitable consequence of ligand-induced perturbations of the probability distribution that represents conformational fluctuations in thermal equilibrium. When high resolution structural data of a protein in empty and ligand-bound forms are available, the COREX algorithm can provide, in principle, an excellent bridge between the energetics of substates distribution in the protein ensemble and structural coordinates. Here we propose a COREX-based strategic approach to link structural perturbations and the free energy changes of allosteric coupling. This strategy might be broadly useful in the endeavor of predicting how specific ligands allosterically regulate the function of specific proteins.

Guanine nucleotide exchange-independent activation of Gs protein by beta2-adrenoceptor.

beta2-adrenoceptor-mediated activation of Gs and adenylyl cyclase or other receptor-mediated G protein activations is believed to occur by receptor-catalyzed replacement of GDP with GTP on the alpha-subunit of the G protein. Here we showed that a beta2-adrenoceptor-Gs system, heterologously expressed in cyc- or human embryonic kidney (HEK)-293 cells, can be activated in the presence of GDP or its phosphorylation-resistant analog, guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). The potency and maximal ability of GDP to activate Gs and adenylyl cyclase were identical to those of GTP. GDP-mediated activation of adenylyl cyclase, similar to that mediated by GTP, was concentration-dependent, required high magnesium concentrations, was inhibited by inverse agonists, and was correlated with the efficacy of receptor ligands used to stimulate the receptor. UDP did not block the GDP-mediated activation, although it completely blocked the formation of a small amount of GTP ( approximately 5% GDP) from GDP. Moreover, the activation of Gs in the presence of GDP was insensitive to cholera toxin treatment of the cells, whereas that observed in the presence of GTP was amplified by the treatment, which showed that the activation observed in the presence of GDP was not mediated by GTP. Therefore, we concluded that GDP itself could mediate beta-adrenoceptor-induced activation of Gs-adenylyl cyclase system as much as GTP. We discuss the results in the context of the current paradigm of receptor-mediated G protein activation and propose an additional mode of activation for beta2-adrenoceptor-G(s) adenylyl cyclase system where nucleotide exchange is not necessary and GDP and GTP play identical roles in receptor-induced Gs protein activation.

Partial rescue of functional interactions of a nonpalmitoylated mutant of the G-protein G alpha s by fusion to the beta-adrenergic receptor.

Most heterotrimeric G-protein alpha subunits are posttranslationally modified by palmitoylation, a reversible process that is dynamically regulated. We analyzed the effects of Galpha(s) palmitoylation for its intracellular distribution and ability to couple to the beta-adrenergic receptor (betaAR) and stimulate adenylyl cyclase. Subcellular fractionation and immunofluorescence microscopy of stably transfected cyc(-) cells, which lack endogenous Galpha(s), showed that wild-type Galpha(s) was predominantly localized at the plasma membrane, but the mutant C3A-Galpha(s), which does not incorporate [(3)H]palmitate, was mostly associated with intracellular membranes. In agreement with this mislocalization, C3A-Galpha(s) showed neither isoproterenol- or GTPgammaS-stimulated adenylyl cyclase activation nor GTPgammaS-sensitive high-affinity agonist binding, all of which were present in the wild-type Galpha(s) expressing cells. Fusion of C3A-Galpha(s) with the betaAR [betaAR-(C3A)Galpha(s)] partially rescued its ability to induce high-affinity agonist binding and to stimulate adenylyl cyclase activity after isoproterenol or GTPgammaS treatment. In comparison to results with the WT-Galpha(s) and betaAR (betaAR-Galpha(s)) fusion protein, the betaAR-(C3A)Galpha(s) fusion protein was about half as efficient at coupling to the receptor and effector. Chemical depalmitoylation by hydroxylamine of membranes expressing betaAR-Galpha(s) reduced the high-affinity agonist binding and adenylyl cyclase activation to a similar degree as that observed in betaAR-(C3A)Galpha(s) expressing membranes. Altogether, these findings indicate that palmitoylation ensured proper localization of Galpha(s) and facilitated bimolecular interactions of Galpha(s) with the betaAR and adenylyl cyclase.

Agonist-directed trafficking explaining the difference between response pattern of naratriptan and sumatriptan in rabbit common carotid artery.

1. Sumatriptan or eletriptan produced vasocontraction in common carotid artery (CCA) by stimulating 5HT(1B) receptors (see also Akin & Gurdal, this issue). 2. Naratriptan as a 5HT(1B/D) agonist, was unable to produce vasocontraction in this artery, but inhibited the vasocontractile response induced by sumatriptan or eletriptan. 3. All these agonists inhibited forskolin-stimulated cyclic AMP production with comparable potencies and maximal responses. This inhibition was mediated by 5HT(1B) receptors: 5HT(1B) antagonist SB216641 (1 microM) completeley antagonized sumatriptan-, eletriptan- or naratriptan-induced cyclic AMP inhibition, but 5HT(1D) antagonist BRL15572 (1 microM) did not affect this response. 4. Naratriptan-induced stimulation of 5-HT(1B) receptors resulted only in adenylate cyclase inhibition, whereas stimulation of these receptors by sumatriptan or eletriptan produced vasocontraction as well. Hence, we concluded that the 5HT(1B)-mediated inhibition of adenylate cyclase was not a sufficient condition to couple the receptor stimulation to vasocontraction. 5. We discussed agonist-induced trafficking as a plausible mechanism for the observed phenomenon.