Ligands for Fluorescence Correlation Spectroscopy on G Protein-Coupled Receptors

Jakobs, Daniel; Sorkalla, Thomas; Häberlein, Hanns

doi:10.2174/092986712803341476

Cited by 11 publications

(6 citation statements)

References 34 publications

(38 reference statements)

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“…Fluorescence is one of the most sensitive spectroscopic methods and many fluorescent ligands have been reported for locating G-protein-coupled receptors (GPCRs), for studying ligand/receptor interactions, and more generally for better understanding their pharmacology and physiological process. − The history of fluorescent ligands is linked to the development of commercially available fluorophores. Organic dyes have been designed and synthesized to exhibit excitation and emission wavelengths, which are compatible with biological observation and are associated to ligands in conjugation reactions.…”

mentioning

confidence: 99%

Original Design of Fluorescent Ligands by Fusing BODIPY and Melatonin Neurohormone

Thireau

Marteaux

Delagrange

et al. 2013

ACS Med. Chem. Lett.

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An original design and synthesis of fluorescent ligands for melatonin receptor studies is presented and consists in the fusion of the endogenous ligand with the fluorescent BODIPY core. Probes I−IV show high affinities for MT 1 and MT 2 melatonin receptors and exhibit fluorescence properties compatible with cell observation.KEYWORDS: GPCRs, melatonin, fluorescence, molecular probes, BODIPY F luorescence is one of the most sensitive spectroscopic methods and many fluorescent ligands have been reported for locating G-protein-coupled receptors (GPCRs), for studying ligand/receptor interactions, and more generally for better understanding their pharmacology and physiological process. 1−4 The history of fluorescent ligands is linked to the development of commercially available fluorophores. Organic dyes have been designed and synthesized to exhibit excitation and emission wavelengths, which are compatible with biological observation and are associated to ligands in conjugation reactions. The addition of such a distinct fluorescent molecule may alter both the chemical (while remaining within a spectrum of lipophilicity to hydrophilicity) and pharmacological (affinity, functionality, etc.) properties of the resulting fluorescent ligand that will modify its cellular behavior.The melatonin receptors MT 1 and MT 2 are members of the GPCR family. They are involved in the regulation of the circadian rhythm and seasonal functions in mammals. They are also implicated in many biological processes ranging from antiinflammatory to antioxidant effects including anti-Parkinson effects 5,6 and were recently reported as part of the mechanism of action of the antidepressant agomelatine, an MT 1 and MT 2 receptor agonist and 5-HT 2C antagonist. 7,8 Despite the discovery of the high affinity agonist and nonselective 2-[ 125 I]-MLT radioligand 9 research on the pharmacology and the functionality/physiological impact of melatonin receptors suffers from the lack of selective probes for these receptors due to their very low level of expression. Moreover, the main disadvantages of this method are the radioactive hazards and the limitations of studying the molecular dynamics of receptor activation. To offer alternative probes, we have developed a concept aiming at using the aromatic core of an endogenous ligand as the source of fluorescence after slight chemical modification and without loss of biological activity. Herein, we report the design and synthesis of fluorescent ligands for melatonin receptor studies thanks to the fusion of the endogenous ligand with the fluorescent BODIPY core.Melatonin presents in its chemical structure an indole ring possessing fluorescent properties that are unfortunately inappropriate for biological analysis due to interferences from other biochromophores (such as tryptophan). 10,11 Our expertise on the melatonin structure/activity relationship 12−16 prompts us to investigate the extension of the π-conjugation of the indole scaffold at position C-2 in order to obtain biologically compatible photophys...

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mentioning

confidence: 99%

Original Design of Fluorescent Ligands by Fusing BODIPY and Melatonin Neurohormone

Thireau

Marteaux

Delagrange

et al. 2013

ACS Med. Chem. Lett.

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“…FCS has been used to measure the diffusion and size of nucleic acids, lipids, individual proteins, and protein complexes in live cells. Recent examples of processes investigated with FCS/ FCCS include viral capsid assembly, mRNA diffusion, enzyme subunit dissociation, system wide measurements of protein diffusion in yeast, gene expression profiles, protein-lipid interactions, and receptor-ligand interactions [52][53][54][55][56][57][58][59][60]. FCCS can be used to quantify and distinguish different populations of receptors, protein complexes, protein-lipid interactions, and protein-DNA interactions [61][62][63][64][65][66][67][68][69].…”

Section: Fluorescence Correlation Spectroscopy (Fcs)mentioning

confidence: 99%

Fluorescence Fluctuation Techniques for the Investigation of Structure-Function Relationships of G-Protein-Coupled Receptors

Youker¹,

Voet²

2020

Fluorescence Methods for Investigation of Living Cells and Microorganisms

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G-protein-coupled receptors (GPCRs) are seven transmembrane receptors that form the largest superfamily of signaling proteins, and the family members function in a diverse array of metabolic pathways including cardiac function, immune response, neurotransmission, smell, taste, cell differentiation and growth, and vision. It is becoming clear that alteration in the quaternary structure of the GPCR receptor can have a profound impact on signaling capabilities. Biochemical, biophysical, physiological, x-ray crystallographic, and computational methods have been used over the last 40-50 years to study the structure and function of GPCRs. Evidence from these studies confirm that GPCRs can be organized as monomers, dimers, and higher-order oligomers. However, many times, these methods require extraction of the receptor from its native environment and high levels of expression and only provide a snapshot of information. A need arose for techniques that could measure the assembly and disassembly of receptors at few-to-single molecule resolution in their native environment at fast time scales. In the last 20 years, fluorescence fluctuation techniques have filled this need and provided new insight into the dynamics of GPCR organization in the absence and presence of ligands, agonists, and antagonists. In this book chapter, we provide a brief introduction to GPCR structure and function [Section 1]. An overview of the theoretical basis for fluorescence fluctuations techniques (FFTs) and how FFTs can be used to study the oligomeric structure of GPCRs in live and fixed cells is explained [Section 2]. We discuss the advantages and limitations of FFTs [Section 3], and finally, we summarize select case studies on GPCR structure and function revealed by FFT experiments [Section 4].

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“…Fluorescent GPCR ligands have been used for studies ranging from localization of receptors in tissues and cells (including an early demonstration of the internalization of ligand-bound receptors in cells [53]), to simple binding assays (in many cases, as replacements for radioligands), to sophisticated probing of the geometry and mechanisms of ligand-receptor interactions and receptor-receptor interactions. Several previous reviews have provided compendia of fluorescent ligands for GPCRs that have been reported in the literature [29, 55–61]. Table 1 presents an updated list of published fluorescent ligands for GPCRs, including information from these previous reviews.…”

Section: Fluorescent Ligandsmentioning

confidence: 99%

Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors

Sridharan

Zuber

Connelly

et al. 2014

Biochimica et Biophysica Acta (BBA) - Biomembranes

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G Protein Coupled Receptors (GPCRs) are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remains unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes, that can be difficult to extract from x-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of GPCRs and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in GPCRs.

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Ligands for Fluorescence Correlation Spectroscopy on G Protein-Coupled Receptors

Cited by 11 publications

References 34 publications

Original Design of Fluorescent Ligands by Fusing BODIPY and Melatonin Neurohormone

Original Design of Fluorescent Ligands by Fusing BODIPY and Melatonin Neurohormone

Fluorescence Fluctuation Techniques for the Investigation of Structure-Function Relationships of G-Protein-Coupled Receptors

Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors

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