Malcolm Rivera
Receptors are chemical structures composed of protein that receive and transmit signals that may be integrated into biological systems. Constitutive activity is defined as ligand-independent activity, resulting in the production of a second messenger in the absence of an agonist. The structural basis of constitutive activity remains largely unexplored, but it is a real pharmacological phenomenon. To test if a receptor is constitutively active, one can use biosensors to detect the constitutive activity of human receptors and the inverse agonist effects of corresponding ligands. Another method is to examine responses to ligand addition, demonstrating that the receptor is functional.
| Characteristics | Values |
|---|---|
| Definition | Constitutive (basal) activity is defined as ligand-independent activity, resulting in the production of a second messenger in the absence of an agonist. |
| Structural Basis | The structural basis of constitutive activity remains largely unexplored. |
| Testing | To test for constitutive activity, one can use biosensors, direct measurements of receptors, or examine responses to ligand addition. |
| Inverse Agonists | Inverse agonists reduce the activity of receptors by inhibiting their constitutive activity (negative efficacy). |
| Mutations | Mutations in receptors can result in increased constitutive activity and have been linked to diseases such as precocious puberty, hyperthyroidism, and night blindness. |
| G-protein-coupled receptors | Many G-protein-coupled receptors show constitutive activity, and mutations in any part of the receptor can cause constitutive activation. |
| Neurotransmitter | The endogenous agonist ligand neurotensin (NTS) is a neurotransmitter and hormone that displays a wide range of biological activities, including cancer cell growth and the pathogenesis of schizophrenia. |
| Therapeutic Relevance | Constitutive GPCR activity is of therapeutic importance, with drugs classified as classical receptor antagonists demonstrating the ability to inhibit ligand-independent activity. |
| Intracellular Receptors | Intracellular receptors are found inside the cell and include cytoplasmic and nuclear receptors. |
| Ligands | Ligands can be proteins, peptides, neurotransmitters, hormones, pharmaceutical drugs, toxins, or other small molecules. |
| DNA Sequencing | Mutations that constitutively activate a receptor can be identified through DNA sequencing of the gene on genomic clones or mRNAs encoding the protein. |
| IHC Testing | IHC is used to identify clinically relevant receptors, but proper controls should be employed to detect false positives and negatives. |
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What You'll Learn
Use bioSensAll® biosensors to detect constitutive activity
Constitutive receptor activity is a real pharmacological phenomenon, and the structural basis of this activity is an area of ongoing research. G protein-coupled receptors (GPCRs) are highly dynamic and versatile signalling molecules that mediate second messenger responses within the cell. GPCRs are a type of cell surface receptor, which are chemical structures composed of protein that receive and transduce signals to be integrated into biological systems.
The bioSensAll® platform offers biosensors that can be used to detect constitutive activity. These biosensors are constructed using different strategies and can be broadly divided into functional categories. The G alpha/gamma-based G protein activation biosensors (GABY) are multimolecular sensors designed to monitor conformational changes within the heterotrimeric G protein complex upon Gα activation and effector interaction. They can be used to detect constitutive activity by measuring the specific activation of each Gα subtype.
The G alpha plasma membrane (GAPL) biosensors are another type of multimolecular sensor that detects plasma membrane recruitment of an effector protein that interacts with Gα subunits upon G protein activation. These biosensors can also measure the specific activation of each Gα subtype, indicating the presence of constitutive activity.
Beta-arrestin membrane recruitment biosensors are another type of multimolecular sensor that detects the recruitment of ß-arrestin 1 or ß-arrestin 2 to the plasma membrane following receptor engagement. These biosensors can be used to study the constitutive activity of receptors by analysing the recruitment and activation of specific proteins.
Additionally, the Diacylglycerol (DAG) biosensor is a multimolecular biosensor that detects the generation of DAG at the plasma membrane following receptor activation. This biosensor can be employed to investigate constitutive activity by examining the generation of DAG, which is indicative of receptor activation.
The bioSensAll® biosensors provide a range of tools to detect and analyse constitutive receptor activity, contributing to our understanding of pharmacological phenomena and their potential therapeutic applications.
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Identify clinically relevant receptors with IHC testing
Immunohistochemistry (IHC) is a diagnostic technique conducted in a laboratory. It involves performing special tests on a patient's biopsy tissue sample to help diagnose a disease more precisely. IHC is particularly useful in diagnosing certain types of cancer, providing the care team with a wealth of information about the disease to help determine treatment.
IHC is routinely used to help diagnose most types of breast cancer, including invasive, metastatic, and recurrent breast cancer. In patients with breast cancer, immunohistochemistry is used to test for hormone receptor status and HER2 status. IHC tests may detect the presence or absence of hormone receptors on breast cancer cells. This knowledge informs how the cancer may be treated, as breast cancers that carry these receptors can be treated with hormone therapy drugs. IHC tests may also check for HER2 receptors to determine whether breast cancer is HER2-positive or HER2-negative. If these receptors are found, the cancer is HER2-positive, which indicates that it’s likely to be fast-growing and may be treated by targeted therapy drugs that block the effects of the HER2 protein.
To perform an IHC test, the pathologist starts by considering the type of cancer suggested by previous tests and then selects an antibody (or several) that might help answer the remaining questions. For example, if the sample is known to contain breast cancer cells but the pathologist needs to find out if the cells are hormone receptor-positive, they will use an estrogen or progesterone antibody that is known to bind with these hormone receptors. Along with the antibody, the pathologist may add chemicals or dyes to the solution. The dyes change the color of cells that contain hormone receptors, making them easily identifiable. The pathologist will then dip a slice of the sample into the antibody solution and place it under a microscope to examine. Once the analysis is complete, a pathology report will be prepared to summarize the diagnosis and other relevant information.
IHC can also be used to improve the accuracy of immunohistochemical estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers.
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Observe structural instability in constitutively active receptors
The structural instability of constitutively active receptors can be observed through various methods, each providing valuable insights into the behaviour of these receptors.
One approach is to examine the rate of denaturation of the purified receptor at elevated temperatures. For example, in the case of the constitutively active mutant of the beta2 adrenergic receptor (CAM), the receptor exhibited a 4-fold increase in the rate of denaturation at 37°C compared to the wild-type receptor. This accelerated denaturation indicates a higher degree of structural instability in the constitutively active form.
Spectroscopic analysis is another powerful technique employed to study structural instability. By utilising conformationally sensitive fluorophores, such as N,N'dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, researchers can observe the structural changes elicited by both agonists and antagonists. This analysis revealed that CAM undergoes more profound structural alterations compared to the wild-type protein, further highlighting its structural instability.
Site-selective fluorescent labelling has also been utilised to map agonist-induced conformational changes in the beta2 adrenergic receptor. This technique helps visualise the structural transitions that occur upon ligand binding, providing a clearer understanding of the dynamic nature of these receptors.
Additionally, molecular dynamics simulations and crystal structure analyses have been employed to study the structure and dynamics of constitutively active neurotensin receptors (NTSR1). These simulations revealed strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face, contributing to robust signalling in cells.
Furthermore, sequence analyses and site-directed mutagenesis studies have provided insights into the structural instability of constitutively active G-protein-coupled receptors (GPCRs). For instance, mutations in certain domains, such as the W6.48 residue, have been shown to strongly impact signalling in some receptors while having minimal effects in others.
While the specific mechanisms underlying the structural instability of constitutively active receptors remain partially unknown, these experimental approaches collectively offer valuable tools to enhance our understanding of their behaviour and facilitate advancements in drug discovery and therapeutic interventions.
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Understand the role of inverse agonists
Inverse agonists are drugs that bind to the same receptor as an agonist but induce a pharmacological response opposite to that of the agonist. A prerequisite for an inverse agonist response is that the receptor must have a constitutive (or intrinsic/basal) level of activity in the absence of any ligand. Inverse agonists decrease the activity of receptors below the basal level.
An agonist increases the activity of a receptor above its basal level. When an agonist binds, an increase in the effect of the receptor is observed because it is in the “on” state. When an antagonist is administered, the receptor is in the “off” state, and no effect is observed. On the other hand, constitutively active receptors are more like dimmer switches: when an agonist is administered, the action a receptor exerts increases. When an inverse agonist is administered, the effects decrease from basal tones.
Inverse agonists have been identified for several receptors, including GABAA, melanocortin, mu opioid, histamine, and beta adrenergic receptors. For example, beta-blockers like carvedilol and bucindolol are low-level inverse agonists at beta adrenoceptors. Histamine is a crucial endogenous transmitter in the body, mediating wakefulness, cognitive ability, and modulating immune cells and appetite. Histamine also mediates allergic responses and can increase secretions of gastric acid. Histaminic receptors are constitutively active, and H1 antihistamine agents are classified as "inverse agonists". Diphenhydramine, for example, is an H1 antihistamine that acts as an inverse agonist at H1 receptors.
Inverse agonists have therapeutic significance and can be used to selectively manipulate the constitutional activity of certain receptors. For instance, treatment with inverse agonists upregulates the surface δ receptors. Inverse agonists also play a role in opioid dependence, as the basal activity at μ receptors may be a contributing factor. In addition, inverse agonism may be relevant in cardiovascular hemodynamics, facilitating the selection of β-blockers in CHF. Constitutive activity in α1- and α2-ARs has also been observed, and activation of both G i and G s was enhanced for CAM receptors.
Constitutive receptor activity and inverse agonism are real pharmacological phenomena, and there is ongoing research into their potential clinical applications. For example, the anti-obesity drugs rimonabant and taranabant are inverse agonists at the cannabinoid CB1 receptor and produced significant weight loss. However, they were withdrawn due to a high incidence of depression and suicidal ideation.
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Study the neurotensin receptor 1 (NTSR1)
Neurotensin Receptor 1 (NTSR1) is a G-protein-coupled receptor that mediates the multiple functions of neurotensin, an endogenous neuromodulator of dopamine transmission. It is a high-affinity receptor, primarily activated by neurotensin (NT). NT is a brain and gastrointestinal peptide that acts on three subtypes of neurotensin receptors: the G-protein-coupled receptors NTS1 (high affinity) and NTS2 (low affinity), and the single transmembrane receptor NTS3 (Sortilin 1).
NTSR1 plays a significant role in various physiological processes, including modulation of immune responses and mucosal healing in conditions like inflammatory bowel disease. It also mediates functions such as hypotension, hyperglycemia, hypothermia, antinociception, and regulation of intestinal motility and secretion.
To study the NTSR1 receptor, researchers have used techniques such as 19F-NMR, hydrogen-deuterium exchange mass spectrometry, and stopped-flow fluorescence spectroscopy to investigate the kinetic mechanism of neurotensin recognition by the receptor. They have also reported the 3.3 Å crystal structure of a constitutively active, agonist-bound NTSR1 and conducted molecular dynamics simulations to understand the mechanistic aspects of constitutive activity.
The structural dynamics of the NTSR1 receptor have been explored, revealing the importance of the ligand-binding pocket and connector residues in the receptor's function. The loss of correlation between these residues and altered receptor dynamics may explain the reduced neurotensin efficacy in constitutively active NTSR1.
Additionally, the role of NTSR1 in various disease states has been investigated. For example, increased NTSR1 expression has been observed in the human colonic mucosa with ulcerative colitis, and NTSR1 has been implicated in the pathogenesis of inflammatory bowel disease and colitis-associated neoplasia. Furthermore, studies have shown that maternal separation alters the NTSR1 gene in the amygdala, impacting neurotransmitter activity and anxiety-like behaviors.
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Frequently asked questions
Constitutive activity is the ability of a receptor to adopt an active “signalling” conformation in a ligand-independent fashion. In other words, it is the ability of a receptor to produce a biological response in the absence of a bound ligand.
To identify a constitutively active receptor, you can use methods such as bioSensAll® biosensors, IHC, direct measurements of receptors, or DNA sequencing of the gene on genomic clones.
Mutations in any part of the receptor can cause constitutive activation. These mutations can be identified through DNA sequencing. Specifically, mutations that remove some stabilizing conformational constraints can make a receptor constitutively active by allowing it to more readily undergo transitions between the inactive and active states.
