The tail wags the dog: possible mechanism for reverse allosteric control of ligand-activated channels.

In this issue of the British Journal of Pharmacology, a new article by Kozuska et al. discusses the multiple salt bridges in the intracellular domain of the 5HT3A receptor. These interactions increase the overall rigidity of the receptor, stabilize its low conducting state and affect the ligand cooperativity. The authors suggest that the allosteric effects of these regions on the receptor may be involved in a possible 'reverse' allosteric modulation of 5HT3 receptors.

What systems can and can't do.

This commentary discusses a paper in this issue by Dr Jillian Baker on the antagonism of histamine H(2) receptors. It is an excellent example of the use of pharmacological principles to determine what systems can and can't do from the point of view of agonist-dependent antagonism. The most common model of antagonism, namely orthosteric, cannot discern agonist type; i.e. all agonists are blocked equally by a given orthosteric antagonist. Therefore, if quantitative assessment of antagonism unveils agonist dependence, then this is something an orthosteric mechanism cannot do and another mechanism must be considered. A simple alternative is a permissive allosteric model whereby the agonist and antagonist interact through conformational changes in the receptor protein. Under these circumstances, an agonist-antagonist dialogue can ensue whereby the nature of the agonist determines the magnitude of antagonist effect. Jillian Baker contrasts antagonist systems with historical data obtained for beta-adrenoceptors and the present data for histamine H(2) receptors where the simpler model of orthosteric antagonism suffices and thus shows how quantitative receptor pharmacology can be used to determine the molecular mechanism of antagonism.

Pharmacological onomastics: what's in a name?

Drugs are named for their primary receptor target and overt action (agonism, antagonism) but the observation of multiple or collateral efficacies emanating from drugs activating a single receptor target is posing a challenge for drug classification and nomenclature. With increasing abilities to detect alteration in cellular function has come the identification of efficacies that are not necessarily manifest in obvious changes in cell response. Specifically, some agonists selectively activate cellular pathways, demonstrate phenotypic behaviour associated with cell type and some antagonists actively induce receptor internalization without activation. In addition, the effects of allosteric modulators can be linked to the nature of the co-binding ligand posing a similar complication in classification and naming. Thus, accurate labels for this new generation of selective drugs may require identification of receptor partners (G-protein type, beta-arrestin) or pathway or, in the case of allosteric modulators, identification of co-binding ligands. The association of distinct phenotypic behaviours with molecules opens the opportunity to better associate clinical effects with distinct pharmacological properties.

Quantitation in receptor pharmacology.

The activity of drugs on receptors can be visualized in appropriate systems. However, for comparisons of activity to be made, these effects must be quantified. The common currency for this quantitation is the dose-response curve. Thus, the shape, location (along the concentration axis), and maximal asymptote of dose-response curves are used to quantify drug activity and comparisons to mathematical models of receptors are made to describe mechanism of action. This review cites examples of where these quantitative procedure yield information beyond what is readily apparent through observation of the data and thus support the use of quantitative methods to maximize the information gained from experiments.

Inverse, protean, and ligand-selective agonism: matters of receptor conformation.

Concepts regarding the mechanisms by which drugs activate receptors to produce physiological response have progressed beyond considering the receptor as a simple on-off switch. Current evidence suggests that the idea that agonists produce only varying degrees of receptor activation is obsolete and must be reconciled with data to show that agonist efficacy has texture as well as magnitude. Thus, agonists can block system constitutive response (inverse agonists), behave as positive and inverse agonists on the same receptor (protean agonists), and differ in the stimulus pattern they produce in physiological systems (ligand-selective agonists). The molecular mechanism for this seemingly diverse array of activities is the same, namely, the selective microaffinity of ligands for different conformational states of the receptor. This paper reviews evidence for the existence of the various types of agonism and the potential therapeutic utility of different agonist types.-Kenakin, T. Inverse, protean, and ligand-selective agonism: matters of receptor conformation.

Uncovering biases in high throughput screens of G-protein coupled receptors.

The ability of high throughput membrane binding assays to detect ligands for G-protein coupled receptors was examined using mathematical models. Membrane assay models were developed using the extended ternary complex model (Samama et al., 1993) as a basis. Ligand binding to whole cells was modeled by adding a G-protein activation step. Results show that inverse agonists bind more slowly and with a lower affinity to receptors in the membrane binding assay than to receptors in whole cells, causing the membrane assay to miss pharmaceutically important inverse agonists. Assay modifications to allow detection of inverse agonists are discussed. Finally, kinetic binding data are shown to provide information about ligand efficacy. This work demonstrates the utility of mathematical modeling in detecting biases in drug-screening assay, and also in suggesting techniques to correct those biases.

The use of stimulus-biased assay systems to detect agonist-specific receptor active states: implications for the trafficking of receptor stimulus by agonists.

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 contrast, functional studies did show differences between the host cell lines and Galpha-subunit enriched cell lines. The relative potency of eight calcitonin agonists was measured in studies of calcium fluorescence in transfected cells containing human calcitonin receptor type 2 by comparing pEC(50) (-log molar concentration producing half-maximal response) values. In Galphas-enriched cells, the relative order of potency of the agonists changed. The host-cell dependent differences in potency ratios ranged from 2-fold to more than 46-fold. This finding is not consistent with the idea that all of the agonists produce response in the same manner (i.e., through a common active state of the receptor). These data are consistent with the idea that these different agonists produce arrays of active states that differentially use G proteins. This idea is discussed in terms of the design of stimulus-bias assay systems to detect agonist-selective receptor active states with resulting potential for increased selectivity of agonists.

Use of constitutive G protein-coupled receptor activity for drug discovery.

This article describes the behavior of transiently transfected human receptors into melanophores and the potential use of constitutive receptor activity to screen for new drug entities. Specifically, transient transfection of melanophores with different concentrations of receptor cDNA presumably leads to increased levels of receptor expression. This leads to an increased response to agonists (both maxima and potency) and, in some cases, an agonist-independent constitutive receptor activity. Transfections with increasing concentrations of the G(s) protein-coupled human calcitonin receptor type 2 (hCTR2) cDNA produced sufficient levels of constitutively activated receptor to cause elevated basal cellular responses. This was observed as a decrease in the transmittance of light through melanophores (consistent with G(s) protein activation) and increased response to human calcitonin. The receptor-mediated nature of this response was confirmed by its reversal with the hCTR2 peptide inverse agonist AC512. A collection of ligands for hCTR2 either increased or decreased constitutive hCTR2 activity, suggesting that the constitutive system was a sensitive discriminator of positive and negative ligand efficacy. Similar results were obtained with G(i)-protein-coupled receptors. Transient transfection of NPY1, NPY2, NPY4, CXCR4, and CCR5 cDNA produced increased light transmittance through melanophores (consistent with G(i)-protein activation). NPY1 cDNA produced little constitutive response on transfection, whereas maximal levels of constitutive activity ranging from 30 to 45% were observed for the other G(i)-protein-coupled receptors. Responses to agonists for these receptors increased (both maxima and potency) with increasing cDNA transfection. The receptor/G(i)-protein nature of both the constitutive and agonist-mediated responses was confirmed by elimination with pertussis toxin pretreatment. These data are discussed in terms of the theoretical aspects of constitutive receptor activity and the applicability of this approach for the general screening of G protein-coupled orphan receptors.

The relative importance of the time-course of receptor occupancy and response decay on apparent antagonist potency in dynamic assays.

1. The potency of the beta1-adrenoceptor antagonist atenolol was measured as an inhibitor of responses to isoprenaline in guinea-pig left atria. Measurements were made in two ways, firstly, by pre-incubating the atria with a given concentration of atenolol followed by an isoprenaline dose-response curve and, secondly, by measuring the response to isoprenaline followed by addition of atenolol. 2. It was found that the estimation of atenolol potency as an antagonist of beta1-adrenoceptors by these two methods gave divergent results. Specifically, it was found that the isoprenaline-induced increased rate of myocardial relaxation was resistant to receptor blockade. Thus, the rate-limiting step in the relaxation response was dissociated from receptor activation and therefore, could not be used for the measurement of receptor occupancy. 3. In contrast, the positive inotropic response was very responsive to receptor occupancy. However, when atenolol was used to block a steady-state isoprenaline response, there was a complicating depression of basal inotropy after receptor blockade that obfuscated measurement of receptor blockade. 4. In general, these data indicated that the blockade of a steady-state agonist response to measure the potency of an antagonist might in some cases yield erroneous results. These studies indicate some caution in the interpretation of blockade responses in pre-contracted or pre-stimulated pharmacological preparations.

Efficacy in drug receptor theory: outdated concept or under-valued tool?

In classical occupancy receptor theory, efficacy is a dimensionless proportionality constant denoting the power of agonists to produce a pharmacological response. In theoretical terms, it is difficult to separate affinity and efficacy estimates of agonists for receptors, hence questioning the value of clearly flawed estimates of efficacy by conventional methods. In this paper, the use of efficacy estimates, the limitations of the current methods to estimate efficacy, and the types of systems in which serious errors in efficacy estimation would be expected, is discussed. Specifically, in constitutively active receptor systems or in those where the receptor interacts with more than one G protein, there are theoretical objections to the use of relative maximal responses as indicators of intrinsic efficacy of agonists.

Constitutive receptor systems for drug discovery.

This paper discusses the use of constitutively active G-protein-coupled receptor systems for drug discovery. Specifically, the ternary complex model is used to define the two major theoretical advantages of constitutive receptor screening-namely, the ability to detect antagonists as well as agonists directly and the fact that constitutive systems are more sensitive to agonists. In experimental studies, transient transfection of Chinese hamster ovary cyclic AMP response element (CRE) luciferase reporter cells with cDNA for human parathyroid hormone receptor, glucagon receptor, and glucagon-like peptide (GLP-1) receptor showed cDNA concentration-dependent constitutive activity with parathyroid hormone (PTH-1) and glucagon. In contrast, no constitutive activity was observed for GLP-1 receptor, yet responses to GLP-1 indicated that receptor expression had taken place. In another functional system, Xenopus laevi melanophores transfected with cDNA for human calcitonin receptor showed constitutive activity. Nine ligands for the calcitonin receptor either increased or decreased constitutive activity in this assay. The sensitivity of the system to human calcitonin increased with increasing constitutive activity. These data indicate that, for those receptors which naturally produce constitutive activity, screening in this mode could be advantageous over other methods.

Pharmacological characterization of receptor-activity-modifying proteins (RAMPs) and the human calcitonin receptor.

Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMP3, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMP3 coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMP3 and 9.45 +/- 0.07 with RAMP3. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMP3 and 8.2 +/- 0.35 with RAMP3. The finding suggests that RAMP3 might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems.

Protean effects of a natural peptide agonist of the G protein-coupled secretin receptor demonstrated by receptor mutagenesis.

G protein-coupled receptors initiate signaling cascades after associating with heterotrimeric G proteins. This is typically initiated by agonist binding, but can also occur spontaneously, particularly in receptors bearing distinct missense mutations. Two such mutations in the parathyroid hormone receptor are associated with constitutive activity, manifesting clinically as Jansen's metaphyseal chondroplasia. We introduce analogous mutations separately and together into the secretin receptor to explore their impact on another family member. Constructs were expressed transiently in COS cells, and had binding and signaling (cAMP generation) studied. Each construct was processed appropriately to lead to cell surface expression and signaling. Secretin bound to the wild-type receptor with two affinity states recognized, 1% of sites in the high affinity state (Ki = 0.5 +/- 0.1 nM) and 99% in the low affinity state (Ki = 23 +/- 3 nM). Mutant receptor binding best fit a single affinity state, having values for Ki of 5 +/- 1 nM (H156R), 8 +/- 1 nM (T322P) and 6 +/- 1 nM (H156R/T322P), with each of these demonstrating a shift to higher affinity than the predominent low affinity state of the wild-type receptor. Each mutant receptor expressed small to moderate constitutive activity, with basal levels of cAMP activity greater than control (P < .01): H156R, 1.4-fold; T322P, 4.5-fold and H156R/T322P, 6.8-fold. The level of basal activity of even the most active construct was only 15% of the maximal response of wild-type receptor. Although each of the single site mutants responded to secretin by increasing their cAMP levels in a concentration-dependent manner, the dual mutant decreased its cAMP in response to hormone (EC50 = 13 nM). Thus, a natural agonist had become an inverse agonist at this unique construct. Because this could reflect reduced normal coupling with Gs or increased aberrant coupling with Gi, the mechanism was further explored using pertussis toxin and a stable analogue of GTP. Although ligand-binding determinants were retained in the dual receptor mutant, the conformation of this receptor upon secretin binding effected a reduction in its basal coupling with Gs, thereby resulting in inverse agonism.

Recombinant human CXC-chemokine receptor-4 in melanophores are linked to Gi protein: seven transmembrane coreceptors for human immunodeficiency virus entry into cells.

This article describes the transient expression of the CXC chemokine receptor-4 in Xenopus laevis melanophores and the resulting functional assay for the endogenous ligand for this receptor stromal cell-derived factor (SDF)-1alpha. Specifically, it will be shown that SDF-1alpha produces increased light transmittance in transfected cells that is consistent with the activation of Gi protein. This stimulus pathway is further implicated by the abolition of this response after pretreatment of the cells with pertussis toxin, a known method for the inactivation of Gi protein. The fact that SDF-1alpha does not produce responses in nontransfected cells and that treatment of the cells with 12G5, an antibody specific for the CXC chemokine receptor-4, eliminates this response indicates that this ligand produces responses by activation of this receptor in these cells. The possible relevance to human immunodeficiency virus (HIV) entry into cells was explored by observing the effects of SDF-1alpha on HIV-mediated cell fusion. It was found that SDF-1alpha blocked cell-to-cell fusion (as has been previously reported) at concentrations 1200-fold greater than those required to produce Gi protein mediated responses. The implications of the functional assay to screening for new drugs to block HIV-mediated fusion is discussed.