## 4.2.2. Receptor interaction

Author: Timo Hamers

Reviewers: Frank van Belleghem and Ludek Blaha

Learning objectives

You should be able to

- explain the possible effects of compound interference with ion channels.
- explain the possible effects of compound interference with G-protein coupled receptors (GPCRs).
- explain the possible effects of compound interference with enzyme linked receptors.
- explain the possible effects of compound interference with nuclear receptors.
- understand what signalling pathways are and how they can be affected to toxic compounds

Key words: Ion channels, G-protein coupled receptors, enzyme linked receptors, nuclear receptors

Introduction

Receptor proteins specifically bind and respond to endogenous signalling ligands such as hormones, prostaglandins, growth factors, or neurotransmitters, by causing a typical cellular response. Receptor proteins can be located in the cell membrane, in the cytosol, and in the nucleus of a cell. Agonistic receptor ligands activate the receptor protein whereas antagonistic ligands inactivate the receptor and prevent (endogenous) agonists from activating the receptor (Figure 1). Based on the role of the receptor protein, binding by ligands may interfere with:

1. ion channels

2. G-protein coupled receptors

3. enzyme-linked receptors

4. nuclear receptors.

Xenobiotic ligands can interfere with these cellular responses by acting as agonistic or antagonistic ligands.

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Figure 1: Activation by the endogenous ligand of a receptor leads to an effect. An agonistic compound may also activate the receptor and leads in cooperation with the endogenous ligand to an enhanced effect. An antagonistic compound also has binding affinity for the receptor, but cannot activate it. Instead, it prevents the endogenous ligand from binding, and activating the receptor, thereby preventing the effect.

1. Ion channels

Ion channels are transmembrane protein complexes that transport ions across a phospholipid bilayer membrane. Ion channels are especially important in neurotransmission, when stimulating neurotransmitters (e.g. acetylcholine or ACh) bind to the (so-called ionotropic) receptor part of the ion channel and open the ion channel for a very short (i.e. millisecond) period of time. As a result, ions can cross the membrane causing a change in transmembrane potential (Figure 2). On the other hand, receptor-binding by inhibiting neurotransmitters (e.g. gamma-aminobutyric acid or GABA) prevents the opening of ion channels.

Figure 2. The acetylcholine receptor (AChR) is a sodium channel. During neurotransmission from the presynaptic to the postsynaptic cell, binding of the neurotransmitter acetylcholine (ACh) to AChR causes opening of the sodium channel allowing depolarisation of the postsynaptic membrane and propagation of the action potential. Drawn by Evelin Karsten-Meessen.

Compounds interfering with sodium channels, for instance, are neurotoxic compounds (see section on Neurotoxicity). They can either block the ion channels or keep them in a prolonged or permanently open state. Many compounds known to interfere with ion channels are natural toxins. For instance, tetrodotoxin (TTX), which is produced by marine bacteria and highly accumulated in puffer fish, and saxitoxin, which is produced by dinoflagellates and is accumulated in shellfish are capable of blocking voltage-gated sodium channels in nerve cells. In contrast, ciguatoxin, which is another persistent toxin produced by dinoflagellates that accumulates in predatory fish positioned high in the food chain, causes prolongation of the opening of voltage-gated sodium channels. Some pesticides like DDT and pyrethroid insecticides also prevent closure of voltage-gated sodium channels in nerve cells. As a consequence, full repolarization of the membrane potential is not achieved. As a consequence, the nerve cells do not reach the resting potential and any new stimulus that would be too low to reach the threshold for depolarization under normal conditions, will now cause a new action potential. In other words, the nerve cells become hyperexcitable and undergo a series of action potentials (repetitive firing) causing tremors and hyperthermia.

2. G-protein coupled receptors (GPCRs)

GPCRs are transmembrane receptors that transfer an extracellular signal into an activated G-protein that is connected to the receptor on the intracellular side of the membrane. G-proteins are heterotrimer proteins consisting of three subunits alpha, beta, and gamma, of which the alpha subunit – in inactivated form – contains a guanosine diphosphate (GDP) molecule. Upon binding by endogenous ligands such as hormones, prostaglandins, or neurotransmitters (i.e. the signal or “first messenger”) to the (so-called metabotropic) receptor, a conformational change in the GPCR complex leads to an exchange of the GDP for a guanosine triphosphate (GTP) molecule in the alpha monomer part of the G-protein, causing release of the activated alpha subunit from the beta/gamma dimer part. The activated alpha monomer can interact with several target enzymes causing an increase in “second messengers” starting signal transduction pathways (see point 3 Enzyme-linked receptors). The remaining beta-gamma complex may also move along the inner membrane surface and affect the activity of other proteins (Figure 3).

Figure 3. Mechanism of GPCR-activation: ligand binding causes a conformational change leading to the release of an activated alpha monomer, which interacts with a target enzyme (causing an increase of second messengers), and a beta-gamma dimer, which may directly affect activity of other proteins (e.g. an ion channel). Source: http://courses.washington.edu/conj/bess/gpcr/gpcr.htm

Two major enzymes that are activated by the alpha monomer are adenylyl cyclase causing an increase in second messenger cyclic AMP (cAMP) and phospholipase C causing an increase in second messenger diacylglycerol (DAG). In turn, cAMP and DAG activate protein kinases, which can phosphorylate many other enzymes. Activated phospholipase C also causes an increase in levels of the second messenger inositol-3-phosphate (I3P), which opens ion channels in the endoplasmic reticulum causing a release of calcium from the endoplasmic store, which also acts as a second messenger. On the other hand, the increase in cytosolic calcium levels is simultaneously tempered by the beta/gamma dimer, which can inhibit voltage-gated calcium channels in the cell membrane. Ultimately, the GPCR signal is extinguished by slow dephosphorylation of GTP into GDP by the activated alpha monomer, causing it to rearrange with the beta/gamma dimer into the original inactivated trimer G-protein (see also https://courses.washington.edu/conj/bess/gpcr/gpcr.htm).

The most well-known example of disruption of GPCR signalling is by cholera toxin (see text block Cholera toxin below).

Despite the recognized importance of GPRCs in medicine and pharmacology, little attention has so-far been paid in toxicology to interaction of xenobiotics with GPCRs. Although a limited number of studies have demonstrated that endocrine disrupting compounds including PAHs, dioxins, phthalates, bisphenol-A, and DDT can interact with GPCR signalling, the toxicological implications of these interactions (especially with respect to disturbed energetic metabolism) remain subject for further research (see review by Le Ferrec and Øvrevik, 2018).

Cholera toxin Cholera toxin is a so-called AB exotoxin by Vibrio cholerae bacteria, consisting of an “active” A-part and a “binding” B-part (see http://www.sumanasinc.com/webcontent/animations/content/diphtheria.html). Upon binding by the B-part to the intestinal epithelium membrane, the entire AB complex is internalized into the cell via endocytosis, and the active A-part is released. This A-part adds an ADP-ribose group to G-proteins making the GTP dephosphorylation of activated G-proteins impossible. As a consequence, activated G-proteins remain in a permanent active state, adenylyl cyclase is permanently activated and cAMP levels rise, which in turn cause an imbalance in ion housekeeping, i.e. an excessive secretion of chloride ions to the gut lumen and a decreased uptake of sodium ions from the gut lumen. Due to the increased osmotic pressure, water is released to the gut lumen causing dehydration and severe diarrhoea (“rice-water stool”).

3. Enzyme-linked receptors

Enzyme-linked receptors are transmembrane receptors that transfer an extracellular signal into an intracellular enzymatic activity. Most enzyme-linked receptors belong to the family of receptor tyrosine kinase (RTK) proteins. Upon binding by endogenous ligands such as hormones, cytokines, or growth factors (i.e. the signal or primary messenger) to the extracellular domain of the receptors, the receptor monomers dimerize and develop kinase activity, i.e. become capable of coupling of a phosphate group donated by a high-energy donor molecule to an acceptor protein. The first substrate for this phosphorylation activity is the dimerized receptor itself, which accepts a phosphate group donated by ATP on its intracellular tyrosine residues. This autophosphorylation is the first step of a signalling pathway consisting of a cascade of subsequent phosphorylation steps of other kinase proteins (i.e. signal transduction), ultimately leading to transcriptional activation of genes followed by a cellular response (Figure 4).

Figure 4. Upon ligand binding, tyrosine kinase receptor (TKR) proteins become autophosphorylated and may phosphorylate (i.e. activate other proteins), including other kinases. Drawn by Evelin Karsten-Meessen.

Xenobiotic compounds can interfere with these signalling pathways in many different ways. Compounds may avoid binding of the endogenous ligand, by blocking the receptor or by chelating the endogenous ligands. Most RTK inhibitors inhibit the kinase activity directly by acting as a competitive inhibitor for ATP binding to the tyrosine residues. Many RTK inhibitors are used in cancer treatment, because RTK overactivity is typical for many types of cancer. This overactivity may for instance be caused by increased levels of receptor-activating growth factors, or to spontaneous dimerization when the receptor is overexpressed or mutated).

4. Nuclear receptors

Nuclear receptors are proteins that are activated by endogenous compounds (often hormones) leading ultimately to expression of genes specifically regulated by these receptors. Apart from ligand binding, activation of most nuclear receptors requires dimerization with a coactivating transcription factor. While some nuclear receptors are located in the nucleus in inactive form (e.g. the thyroid hormone receptor), most nuclear receptors are located in the cytosol, where they are bound to co-repressor proteins (often heat-shock proteins) keeping them in an inactive state. Upon ligand binding to the ligand binding domain (LBD) of the receptor, the co-repressor proteins are released and the receptor either forms a homodimer with a similar activated nuclear receptor or forms a heterodimer with a different nuclear receptor, which is often the retinoid-X receptor (RXR) for nuclear hormone receptors. Before or after dimerization, activated nuclear receptors are translocated to the nucleus. In the nucleus, they bind through their DNA-binding domain (DBD, or “zinc finger”) to a responsive element in the DNA located in the promotor region of receptor-responsive genes. Consequently, these genes are transcribed to mRNA in the nucleus, which is further translated into proteins in the cell cytoplasm, see Figure 5).

Figure 5. Activation of a cytosolic nuclear receptor (NR). Upon ligand binding (e.g. a hormone), the heat shock proteins (HSP) dissociate from the ligand-receptor complex, which forms a heterodimer before entering the nucleus. After recruiting other coactivating transcription factors, the activated dimer binds to the hormone response element (HRE). RNA polymerase binds to this complex and starts transcription of mRNA, which is excreted from the nucleus into the cytosol and transcribed in corresponding proteins. Source: https://upload.wikimedia.org/wikipedia/commons/3/3f/Nuclear_receptor_action.png

Xenobiotic compounds may act as agonist or antagonists of nuclear receptor activation. Chemicals that act as a nuclear receptor agonist mimic the action of the endogenous activator(s), whereas chemicals that act as a nuclear receptor antagonist basically block the LBD of the receptor, preventing the binding of the endogenous activator(s). Over the past decades, interaction of xenobiotics with nuclear receptors involved in signalling of both steroid and non-steroid hormones has gained a lot of attention of researchers investigating endocrine disruption (link to section on Endocrine Disruption). Nuclear receptor activation is also the key mechanism in dioxin-like toxicity (see text block dioxin-like toxicity below).

Dioxin-like toxicity The term dioxins refers to polyhalogenated dibenzo-[p]-dioxin (PHDD) compounds, which are planar molecules consisting of two halogenated aromatic rings, which are connected by two ether bridges. The most potent and well-studied dioxin is 2,3,7,8-tetrachloro-[p]-dibenzodioxin (2,3,7,8-TCDD), which is often too simply referred to as TCDD or even just “dioxin”. Other compounds with similar properties (dioxin-like compounds) include polyhalogenated dibenzo-[p]-furan (PHDF) compounds (often too simply referred to as “furans”), which are planar molecules consisting of two halogenated aromatic rings connected by one ether bridge and one carbon-carbon bond. A third major class of dioxin-like compounds belong to the polyhalogenated biphenyls (PHB), which consist of two halogenated aromatic rings connected only by a carbon-carbon bond. The most well-known compounds belonging to this latter category are the polychlorinated biphenyls (PCBs). Of all PHDD, PHDF or PHB compounds, only the persistent and planar compounds are considered dioxin-like compounds. For the PHBs, this implies that they should contain zero or at maximum one halogen-substitution in any of the four ortho-positions (see examples below). Non-ortho-substituted PHBs can easily obtain a planar confirmation with the two aromatic rings in one planar field, whereas mono-ortho-substituted PHBs can obtain such confirmation at higher energetic costs..
| 2,3,7,8-tetrachlorodibenzo-[p]-dioxin (2,3,7,8-TCDD) is the most potent and well-studied dioxin-like compound, usually too simply referred to as “dioxin”.
| 2,3,7,8-tetrachlorodibenzo-[p]-furan (2,3,7,8-TCDF) a dioxin-like compound equally potent to 2,3,7,8-TCDD. It is usually too simply referred to as “furan”.
| 3,3’,4,4’,5-pentachlorinated biphenyl (PCB-126) is the most potent dioxin-like PCB compound, with no chlorine substitution in any of the four ortho positions next to the carbon-carbon bridge
| 2,3’,4,4’,5-pentachlorinated biphenyl (PCB-118) is a weak dioxin-like PCB compound, with one chlorine substitution in the four ortho positions next to the carbon-carbon bridge
| 2,2’,4,4’,5,5’-hexachlorinated biphenyl (PCB-153) is a non-dioxin-like (NDL) PCB compound, with two chlorine substitution in the four ortho positions next to the carbon-carbon bridge
The planar composition is required for the dioxin-like compounds to fit as a key in the lock of the arylhydrocarbon (AhR) receptor (also known as the “dioxin-receptor or DR), present in the cytosol. The activated AhR then dissociates from its repressor proteins, is translocated to the nucleus, and forms a heterodimer with the AhR nuclear translocator (ARNT). The AhR-ARNT complex binds to dioxin-response elements (DRE) in the promotor regions of dioxin-responsive genes in the DNA, ultimately leading to transcription and translation of these genes (Figure 6). Famous examples of such genes belong to the CYP1, UGT, and GST families, which are Phase I and Phase II metabolic enzymes whose activation by the AhR-ARNT complex is a natural response triggered by the need to remove xenobiotics (link to section on Xenobiotic metabolism and defence). Other genes with a DRE in their promotor region include genes involved in protein phosphorylation, such as the proto-oncogen c-raf and the cyclin dependent kinase inhibitor p27. Figure 6. Classical mechanism of induction of gene expression by compounds interacting with the arylhydrocarbon receptor (AhR). The AhR is present in the cytosol as a complex with two heat shock proteins (hsp90), X-associated protein 2 (XAP2). Upon ligand binding by polyhalogenated aromatic hydrocarbons (see text) the complex is transferred to the nucleus, where the activated AhR first dissociates from its chaperone proteins and then forms a dimer with the AhR nuclear translocator (ARNT). Upon binding of the dimer to dioxin responsive elements (DREs) in the DNA, dioxin-responsive genes (such as cytochrome P-4501A1 or CYP1A1) or transcribed and translated. Redrawn from Denison and Nagy (2003) by by Evelin Karsten-Meessen. This classical mechanism of ligand:AhR:ARNT:DRE complex-dependent induction of gene expression, however, cannot explain all the different types of toxicity observed for dioxins, including immunotoxicity, reproductive toxicity and developmental toxicity. Still, these effects are known to be mediated through the AhR as well, as they were not observed in AhR knockout mice. This can partly be explained by the fact that not all genes that are under transcriptional control of a DRE are known yet. Moreover, AhR dependent mechanisms other than this classical mechanism have been described. For instance, AhR activation may have anti-estrogenic effects because activated AhR (1) binds to the estrogen receptor (ER) and targets it for degradation, (2) binds (with ARNT) to inhibitory DREs in the promotor of ER-dependent genes, and (3) competes with the ER-dimer for common coactivators. Although dioxin-like compounds absolutely require the AhR to exert their major toxicological effects, several AhR independent effects have been described as well, such as AhR-independent alterations in gene expression and changes in Ca2+ influx related to changes in protein kinase activity. Apart from the persistent halogenated dioxinlike compounds described above, other compounds may also activate the AhR, including natural AhR agonists (nAhRAs) found in food (e.g. indolo[3,2-b]carbazole (ICZ) in cruciferous vegetables, bergamottin in grapefruits, tangeretin in citrus fruits), and other planar aromatic compounds, including polycyclic aromatic hydrocarbons (PAHs) produced by incomplete combustion of organic fuels. Upon activation of the AhR, these non-persistent compounds are metabolized by the induced CYP1A biotransformation enzymes. In addition, an endogenous AhR ligand called 6-formylindolo[3,2-b]carbazole (FICZ) has been identified. FICZ is a mediator in many physiological processes, including immune responses, cell growth and differentiation. Endogenous FICZ levels are regulated by a negative feedback FICZ/AhR/CYP1A loop, i.e. FICZ activates AhR and is metabolized by the subsequently induced CYP1A. Dysregulation of this negative feedback loop by other AhR agonists may disrupt FICZ functioning, and could possibly explain some of the effects observed for dioxinlike compounds. Further reading: Denison, M.S., Soshilov, A.A., He, G., De Groot, D.E., Zhao, B. (2011). Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the Aryl hydrocarbon (Dioxin) Receptor. Toxicological Sciences 124, 1-22.

Further reading:

Boelsterli, U.A. (2009). Mechanistic Toxicology (2nd edition). Informa Healthcare, New York, London.

Denison, M.S., Nagy, S.R. (2003). Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annual Reviews of Pharmacology and Toxicology 43, 309–334.

Le Ferrec, E., Øvrevik J. (2018). G-protein coupled receptors (GPCR) and environmental exposure. Consequences for cell metabolism using the b-adrenoceptors as example. Current Opinion in Toxicology 8, 14-19.

https://courses.washington.edu/conj/bess/gpcr/gpcr.htm