Commentry

On the importance of the “antagonist assumption” to how receptors express themselves

Recombinant systems have been used for evaluating the properties of G-protein-coupled receptors (GPCRs) on the assumption of cell surface expression. However, many GPCRs, including muscarinic acetylcholine receptors (mAChRs), have also been reported to be distributed in intracellular organelles in native tissues and cell lines. In this study, we compared the pharmacological profiles of exogenously and endogenously expressed M1-mAChRs, and evaluated the functional properties of these receptors.

Recombinant M1-mAChRs were expressed exogenously in Chinese hamster ovary cells (CHO-M1 cells) and compared with endogenously expressed M1-mAChRs in N1E-115 neuroblastoma cells. The pharmacological and functional profiles were evaluated using cell-permeable antagonists (1-quinuclidinyl-benzilate (QNB), pirenzepine and atropine) and cell-impermeable antagonists (N-methylscopolamine (NMS) or MT-7).

M1-mAChRs were seen at the cell surface and intracellular sites in both cell lines. Under whole cell conditions, intracellular M1-mAChRs were mainly recognized by cell-permeable ligands, but scarcely by cell-impermeable ligands (at less than 100 nM). In CHO-M1 cells, M1-mAChR activation by carbachol resulted in Ca^2 + mobilization, ERK1/2 phosphorylation and a reduction in thymidine incorporation, all of which were completely inhibited by MT-7, indicating the involvement of surface M1-mAChRs. In N1E-115 cells, Ca^2 + mobilization occurred through surface M1-mAChRs, whereas ERK1/2 phosphorylation and acceleration of thymidine incorporation were mediated through intracellular M1-mAChRs.

Exogenous and endogenous M1-mAChRs are present at both the cell surface and the intracellular organelles, and the pharmacological properties of geographically distinct M1-mAChRs are different, and may depend on cell background and/or exogenous or endogenous origin.

[³H]-CGP12177 biphasically bound to β-adrenoceptors with high and low affinities in the segments and crude membranes of rabbit left ventricle. The low-affinity sites for [³H]-CGP12177 in the segments was double in density, compared to the density of high-affinity sites. Total abundance of the β-adrenoceptors decreased to approximately 10% upon tissue homogenization, and the proportion of low-affinity sites was the same as that of the high-affinity sites in the membranes. The majority of the high-affinity binding sites of [³H]-CGP12177 in the segments and the membranes were β_1H-adrenoceptor, being highly sensitive to propranolol and β₁-antagonists (atenolol and ICI-89,406), whereas the low-affinity binding sites showed a β_1L-profile (less sensitive to propranolol and β₁-, β₂-, and β₃-antagonists). Furthermore, a part of the β_1L-adrenoceptors was insensitive to atenolol, ICI-89,406, and/or isoproterenol. The present binding study clearly shows that β_1L-adrenoceptors occur as a distinct phenotype from β_1H-adrenoceptors in rabbit ventricle. However, quantitative imbalance between β_1H- and β_1L-adrenoceptors and divergent ligand–β_1L-adrenoceptor interactions suggest a possibility that the β_1L-adrenoceptor may not reflect a simple conformational change or allosteric state in the β₁-adrenoceptor molecule.

Antagonist affinity measurements have traditionally been considered important in characterizing the cell-surface receptors present in a particular cell or tissue. A central assumption has been that antagonist affinity is constant for a given receptor–antagonist interaction, regardless of the agonist used to stimulate that receptor or the downstream response that is measured. As a consequence, changes in antagonist affinity values have been taken as initial evidence for the presence of novel receptor subtypes. Emerging evidence suggests, however, that receptors can possess multiple binding sites and the same receptor can show different antagonist affinity measurements under distinct experimental conditions. Here, we discuss several mechanisms by which antagonists have different affinities for the same receptor as a consequence of allosterism, coupling to different G proteins, multiple (but non-interacting) receptor sites, and signal-pathway-dependent pharmacology (where the pharmacology observed varies depending on the signalling pathway measured).

A central dogma of G protein-coupled receptor (GPCR) pharmacology has been the concept that unlike agonists, antagonist ligands display equivalent affinities for a given receptor, regardless of the cellular environment in which the affinity is assayed. Indeed, the widespread use of antagonist pharmacology in the classification of receptor expression profiles in vivo has relied upon this ‘antagonist assumption’. However, emerging evidence suggests that the same gene-product may exhibit different antagonist pharmacological profiles, depending upon the cellular context in which it is expressed—so-called ‘phenotypic’ profiles. In this commentary, we review the evidence relating to some specific examples, focusing on adrenergic and muscarinic acetylcholine receptor systems, where GPCR antagonist/inverse agonist pharmacology has been demonstrated to be cell- or tissue-dependent, before going on to examine some of the ways in which the cellular environment might modulate receptor pharmacology. In the majority of cases, the cellular factors responsible for generating phenotypic profiles are unknown, but there is substantial evidence that factors, including post-transcriptional modifications, receptor oligomerization and constitutive receptor activity, can influence GPCR pharmacology and these concepts are discussed in relation to antagonist phenotypic profiles. A better molecular understanding of the impact of cell background on GPCR antagonist pharmacology is likely to provide previously unrealized opportunities to achieve greater specificity in new drug discovery candidates.

The effects of a range of cannabinoid receptor agonists and antagonists on phytohaemagglutinin-induced secretion of interleukin-2 from human peripheral blood mononuclear cells were investigated. The nonselective cannabinoid receptor agonist WIN55212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholinylmethyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate) and the selective cannabinoid CB₂ receptor agonist JWH 015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone) inhibited phytohaemagglutinin (10 μg/ml)-induced release of interleukin-2 in a concentration-dependent manner (IC_1/2max, WIN55212-2=8.8×10⁻⁷ M, 95% confidence limits (C.L.)=2.2×10⁻⁷–3.5×10⁻⁶ M; JWH 015=1.8×10⁻⁶ M, 95% C.L.=1.2×10⁻⁶–2.9×10⁻⁶ M, n=5). The nonselective cannabinoid receptor agonists CP55,940 ((−)-3-[2-hydroxy-4-(1,1-dimethyl-hepthyl)-phenyl]4-[3-hydroxypropyl]cyclo-hexan-1-ol), Δ⁹-tetrahydrocannabinol and the selective cannabinoid CB₁ receptor agonist ACEA (arachidonoyl-2-chloroethylamide) had no significant (P>0.05) inhibitory effect on phytohaemagglutinin-induced release of interleukin-2. Dexamethasone significantly (P<0.05) inhibited phytohaemagglutinin-induced release of interleukin-2 in a concentration-dependent manner (IC_1/2max=1.3×10⁻⁸ M, 95% C.L.=1.4×10⁻⁹–3.2×10⁻⁸ M). The cannabinoid CB₁ receptor antagonist SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride) (10⁻⁶ M) did not antagonise the inhibitory effect of WIN55212-2 whereas the cannabinoid CB₂ receptor antagonist SR144528 (N-(1,S)-endo-1,3,3-trimethyl bicyclo(2,2,1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) antagonised the inhibitory effect of WIN55212-2 (pA₂=6.3±0.1, n=5). In addition, CP55,940 (10⁻⁶ M) and Δ⁹-tetrahydrocannabinol (10⁻⁶ M) also antagonised the inhibitory effects of WIN55212-2 (pA₂=6.1±0.1, n=5 and pA₂=6.9±0.2, n=5). In summary, WIN55,212-2 and JWH 015 inhibited interleukin-2 release from human peripheral blood mononuclear cells via the cannabinoid CB₂ receptor. In contrast, CP55,940 and Δ⁹-tetrahydrocannabinol behaved as partial agonists/antagonists in these cells.

Historically, the ability of a ligand to bind to its receptor and the ability to subsequently activate that receptor have been described as the properties of affinity and intrinsic efficacy, respectively. These properties were originally believed to be independent of one another; both are possessed by ligands classed as “agonists,” and they have served as the quantitative foundation of the drug and receptor classification process. Although affinity has been interpreted readily in physicochemical terms, equivalent molecular models for efficacy remain elusive. In recent times, there has been a significant paradigm shift in our understanding of the interrelationship between affinity and intrinsic efficacy, particularly on theoretical grounds, yet the actual methods available to measure these parameters remain largely operational. Nevertheless, a number of approaches, based on both functional measurements and radioligand binding studies, are available to quantify agonist efficacy on a relative scale and, to date, these remain the most practical. This commentary discusses the most common of these methods, their advantages and limitations, the dependence of the expression of agonism on the chosen assay system, and the impact of recent biochemical and molecular biological advances on the study of efficacy. Additionally, some of the more contemporary theories regarding the molecular nature of efficacy are briefly discussed, as well as the caveats that always must be borne in mind when any determinations of relative agonist efficacy are made.