Tiotidine, a Histamine H2 Receptor Inverse Agonist That Binds with High Affinity to an Inactive G-Protein—Coupled Form of the Receptor. Experimental Support for the Cubic Ternary Complex Model

Abstract

Knowing the importance for research and pharmacological uses of proper ligand classification into agonists, inverse agonists, and antagonists, the aim of this work was to study the behavior of tiotidine, a controversial histamine H2 receptor ligand. We found that tiotidine, described previously as an H2 antagonist, actually behaves as an inverse agonist in U-937 cells, diminishing basal cAMP levels. [3 H]Tiotidine showed two binding sites, one with high affinity and low capacity and the other with low affinity and high capacity. The former site disappeared in the presence of guanosine 5Ј-O-(3-thio)triphosphate, indicating that it belongs to a subset of receptors coupled to G-protein, showing the classic binding profile for an agonist. Considering the occupancy models developed up to now, the only one that explains tiotidine dual behavior is the cubic ternary complex (CTC) model. This model allows Gprotein to interact with the receptor even in the inactive state. We showed by theoretical simulations based on the CTC model of dose-response and binding experiments that tiotidine biases the system to a G-protein-coupled form of the receptor that is unable to evoke a response. This theoretical approach was supported by experimental results in which an unrelated Gprotein-coupled receptor that also signals through G␣ s -protein (␤ 2 -adrenoreceptor) was impeded by tiotidine. This interference clearly implies that tiotidine biases the system to G␣ s -coupled form of the H2 receptor and turns G␣ s -protein less available to interact with ␤ 2 -adrenoreceptor. These findings not only show that tiotidine is an H2 inverse agonist in U-937 cells but also provide experimental support for the CTC model.Receptors coupled to G-proteins (GPCRs) play a major role in signal transduction and are the targets for a large number of therapeutic drugs. Although much is known about signal transduction pathways, the mechanism by which ligands bind to and activate GPCRs remains unclear. In an attempt to understand such mechanisms, several occupancy models have been developed. The first theoretical model of receptor function that included G-proteins was the ternary complex model of De Léan et al. (1980) in which the receptor possesses two binding sites, one for the ligand on the extracellular side and other for the G-protein in the intracellular side. This model is a generalization of former models, in which only the free receptor and the receptor ligand complex existed, allowing

Multiple Signaling States of G-Protein-Coupled Receptors

“…In experimental support of the cubic ternary complex model, tiotidine was found to be an inverse agonist that binds with high affinity to a form of the H2 histamine receptor coupled to Gs that was incapable of signaling. This was documented by showing that in the same cell, tiotidine also impeded the signaling of the ␤ 2 -AR system, that is MULTIPLE ACTIVATION STATES OF GPCRS also coupled to Gs, supposedly by the histamine receptor recruitment of Gs (Monczor et al, 2003).…”

International Union of Basic and Clinical Pharmacology. XCVIII. Histamine Receptors

“…The H 2 receptor was the first histamine receptor subtype for which constitutive activation of adenylyl cyclase has been firmly established for both rat (Smit et al, 1996a) and human variants (Alewijnse et al, 1998(Alewijnse et al, , 2000Monczor et al, 2003). Clinically used H 2 receptor antagonists, including cimetidine (24) and ranitidine (25), behaved as inverse agonists, whereas up to now only burimamide has been described as neutral antagonist (Smit et al, 1996a).…”

“…The H 2 receptor stimulates adenylyl cyclase-mediated cAMP formation in membrane preparations from various tissues as well as cAMP accumulation in various native cells (for review, see Hill, 1990, andHill et al, 1997) and cells expressing recombinant H 2 receptors (Alewijnse et al, 1998(Alewijnse et al, , 2000Monczor et al, 2003). As for many adenylyl cyclase-coupled GPCRs, the H 2 receptor stimulates cAMP production via the coupling to G s proteins, as indicated by, for example, agonistinduced photoaffinity labeling.…”

An 80-Amino Acid Deletion in the Third Intracellular Loop of a Naturally Occurring Human Histamine H3 Isoform Confers Pharmacological Differences and Constitutive Activity

“…Furthermore, we simulated radioligand competition and dose-response curves for the hH 3 R(445) and hH 3 R(365) in a G-protein-dependent manner (Monczor et al, 2003). For the competition curves, we assumed the radioligands and the competing ligands to have properties as described above.…”