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Recombinant roulette versus the apparent virtues of ‘natural’ cell receptor systems: receptor genotypes versus phenotypes

Terpenes are the major constituents of essential oils in plants. In recent years, terpenes have become of clinical relevance due to their ability to suppress cancer development. Their effect on cellular proliferation has made them promising agents in the prevention or treatment of many types of cancer. In the present study, a subset of different monoterpenes was investigated for their molecular effects on the hepatocellular carcinoma cell line Huh7. Using fluorometric calcium imaging, acyclic monoterpene (−)-citronellal was found to induce transient Ca²⁺ signals in Huh7 cells by activating a cAMP-dependent signaling pathway. Moreover, we detected the (−)-citronellal-activated human olfactory receptor OR1A2 at the mRNA and protein levels and demonstrated its potential involvement in (−)-citronellal-induced calcium signaling in Huh7 cells. Furthermore, activation of OR1A2 results in phosphorylation of p38 MAPK and reduced cell proliferation, indicating an effect on hepatocellular carcinoma progression. Here, we provide for the first time data on the molecular mechanism evoked by (−)-citronellal in human hepatocellular carcinoma cells. The identified olfactory receptor could serve as a potential therapeutic target for cancer diagnosis and treatment.

The 5-HT₆ receptor belongs to the G-protein-coupled receptor (GPCR) family. This chapter reviews some reported experimental approaches that can be used for a better functional characterization and classification of 5-HT₆ receptor ligands. The chapter summarizes the results obtained using some representative 5-HT₆ receptor agonists and antagonists. 5-HT₆ receptor functionality is being revealed to be much more complex than initially defined. Based on the existing data and depending on the agonist used, different selective receptor active states that activate different cellular pathways may be achieved. Besides Gs-mediated cAMP formation, other messenger systems may be involved. The full characterization of the functional profile of 5-HT₆ receptor is still pending, and new players in the signaling cascade of 5-HT₆ receptor could take the stage in the near future.

G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization processes and GPCR up- and downregulation. GPCR function can also be regulated by several proteins that directly interact with the receptor and thereby modulate receptor activity. An additional mechanism by which receptor signalling is regulated involves an emerging class of proteins, the so-called regulators of G protein signalling (RGS). In this review we will describe some of these control mechanisms in more detail with some specific examples in the cardiovascular system. In addition, we will provide an overview on RGS proteins and the involvement of RGS proteins in cardiovascular function.

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein αolf and adenylyl cyclase type-III to the Ca²⁺ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Gαolf in odorant-specific signaling out of OR. The employment of the non-typical G protein α15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Gα15 or Gαolf. We finally established an EC₅₀-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.

Ligand-selective receptor conformations introduce the concept of ‘texture’ to drug effects, with respect to ligands possessing quality in addition to quantity of efficacy. This cell-dependent phenotypic efficacy extends to ligand properties beyond G-protein signaling and, in terms of drug development, presents a two-edged sword to pharmacologists. On the one hand, such efficacy promises more selective agonism but on the other hand it predicts problems associated with the use of recombinant or natural lead optimization assays as predictors of therapeutic value in humans. In this article, the evidence to suggest that not all agonists produce the same receptor active state is reviewed.

The assessment of cardiovascular effects of well-recognised ligands for their known receptors can produce misleading results if measurements are restricted to blood pressure and heart rate in anaesthetised animals. Therefore, we propose that the most reliable delineation of any role for a particular ligand–receptor system in cardiovascular regulation can only be achieved by determination of the detailed haemodynamic effects of manipulating the system in vivo, in the absence of anaesthesia. This approach is powerful because, firstly, differences between in vitro and in vivo effects, possibly attributable to disparities between receptor genotype and receptor phenotype, are highlighted; and secondly, regional heterogeneity of cardiovascular effects, possibly in the absence of changes in systemic arterial blood pressure, can be detected.