Photoaffinity Labeling of the Human A<sub>2A</sub> Adenosine Receptor and Cross-link Position Analysis by Mass Spectrometry

Muranaka, Hideyuki; Momose, Takumaro; Handa, Chiaki; Ozawa, Tomonaga

doi:10.1021/acsmedchemlett.7b00138

Cited by 21 publications

(29 citation statements)

References 35 publications

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“…Nowadays, it is sometimes possible to identify the residue captured in the crosslinking reaction using tandem mass spectrometry (MS/MS). However, MS/MS on GPCRs is only possible when large amounts of isolated receptor are available, as it is for instance the case of rhodopsin [ 72 ] or receptors obtained by either yeast [ 73 , 74 , 75 ] or bacterial expression [ 58 ].…”

Section: The Contact Interfacementioning

confidence: 99%

Capturing Peptide–GPCR Interactions and Their Dynamics

Kaiser

Coin

2020

Molecules

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Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

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Section: The Contact Interfacementioning

confidence: 99%

Capturing Peptide–GPCR Interactions and Their Dynamics

Kaiser

Coin

2020

Molecules

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“…Muranaka et al started from the not-so-A 2A R-selective SCH58261 scaffold [ 144 ] and incorporated the trifluoromethyl diazirine group to yield photoaffinity ligand 9 (Fig. 5 , Table 4 ) [ 120 ]. When purified hA 2A R was photolabeled with this ligand and subjected to protease digestion, cross-link positions were identified with LC-MS/MS.…”

Section: Covalent Ligandsmentioning

confidence: 99%

Molecular probes for the human adenosine receptors

Yang

Heitman

IJzerman

et al. 2020

Purinergic Signalling

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Adenosine receptors, G protein–coupled receptors (GPCRs) that are activated by the endogenous ligand adenosine, have been considered potential therapeutic targets in several disorders. To date however, only very few adenosine receptor modulators have made it to the market. Increased understanding of these receptors is required to improve the success rate of adenosine receptor drug discovery. To improve our understanding of receptor structure and function, over the past decades, a diverse array of molecular probes has been developed and applied. These probes, including radioactive or fluorescent moieties, have proven invaluable in GPCR research in general. Specifically for adenosine receptors, the development and application of covalent or reversible probes, whether radiolabeled or fluorescent, have been instrumental in the discovery of new chemical entities, the characterization and interrogation of adenosine receptor subtypes, and the study of adenosine receptor behavior in physiological and pathophysiological conditions. This review summarizes these applications, and also serves as an invitation to walk another mile to further improve probe characteristics and develop additional tags that allow the investigation of adenosine receptors and other GPCRs in even finer detail.

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“…[35] Using the structures of highly potent A 2A AR antagonists 13 and 14,Muranaka and co-workers developed probes 15 and 16 to study its binding site on A 2A AR (Figure 4). [26] After initial assays demonstrated am ore than 30-foldh igher affinity of 16 over 15,o nly probe 16 was used in photolabelinge xperiments. Using humanA 2A AR, they found that 16 had been selectively crosslinked to the amino acid residue Tyr271 in TM7.…”

Section: Human a 2a Adenosine Receptor Antagonist Binding-site Identimentioning

confidence: 99%

Recent Applications of Diazirines in Chemical Proteomics

Halloran

Lumb

2019

Chemistry A European J

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The elucidation of substrate–protein interactions is an important component of the drug development process. Due to the complexity of native cellular environments, elucidating these fundamental biochemical interactions remains challenging. Photoaffinity labeling (PAL) is a versatile technique that can provide insight into ligand‐target interactions. By judicious modification of substrates with a photoreactive group, PAL creates a covalent crosslink between a substrate and its biological target following UV‐irradiation. Among the commonly employed photoreactive groups, diazirines have emerged as the gold standard. In this Minireview, recent developments in the field of diazirine‐based photoaffinity labeling will be discussed, with emphasis being placed on their applications in chemical proteomic studies.

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Photoaffinity Labeling of the Human A_2A Adenosine Receptor and Cross-link Position Analysis by Mass Spectrometry

Cited by 21 publications

References 35 publications

Capturing Peptide–GPCR Interactions and Their Dynamics

Capturing Peptide–GPCR Interactions and Their Dynamics

Molecular probes for the human adenosine receptors

Recent Applications of Diazirines in Chemical Proteomics

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Photoaffinity Labeling of the Human A2A Adenosine Receptor and Cross-link Position Analysis by Mass Spectrometry

Cited by 21 publications

References 35 publications

Capturing Peptide–GPCR Interactions and Their Dynamics

Capturing Peptide–GPCR Interactions and Their Dynamics

Molecular probes for the human adenosine receptors

Recent Applications of Diazirines in Chemical Proteomics

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Product

Resources

About

Photoaffinity Labeling of the Human A_2A Adenosine Receptor and Cross-link Position Analysis by Mass Spectrometry