Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Misu, Ryosuke; Oishi, Shinya; Setsuda, Shohei; Noguchi, Taro; Kaneda, Masato; Ohno, Hiroaki; Evans, Barry J.; Navenot, Jean-Marc; Peiper, Stephen C.; Fujii, Nobutaka

doi:10.1016/j.bmcl.2013.02.098

Cited by 9 publications

(8 citation statements)

References 36 publications

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“…For the Kp-14-based probe 4, a polyethylene glycol (PEG) linker was employed between the fluorophore and the peptide N-terminus to avoid the possibility of reduced binding affinity resulting from the bulky fluorophore group. 23 Several TMR-labeled kisspeptin derivatives were synthesized. The peptide chains for Kp-52 and Kp-14 were constructed using Fmoc-based solid phase peptide synthesis (SPPS) on CLEAR-Amide resin and NovaSyn TGR resin, respectively (Scheme 1).…”

Section: Design and Synthesis Of Kisspeptin-based Fluorescent Probes mentioning

confidence: 99%

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Design and synthesis of fluorescent probes for GPR54

Kaneda

Misu

Ohno

et al. 2014

Bioorganic & Medicinal Chemistry

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Kisspeptins are neuropeptides that induce the secretion of gonadotropin-releasing hormone via the activation of the cognate receptor, G-protein coupled receptor 54 (GPR54). The kisspeptin-GPR54 axis is associated with the onset of puberty and the maintenance of the reproductive system. In this study, several fluorescent probes have been designed and synthesized for rat GPR54 through the modification of the N-terminus of rat kisspeptins to allow for the visualization of the expression and localization of kisspeptin receptor(s) in living cells and native tissues. The tetramethylrhodamine (TMR) and rhodamine green (RG)-labeled kisspeptins exhibited good binding and agonistic activities towards GPR54, and the results of the application studies demonstrated that these fluorescent probes could be used effectively for the detection of GPR54 receptors in flow cytometry and confocal microscopy experiments.

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Section: Design and Synthesis Of Kisspeptin-based Fluorescent Probes mentioning

confidence: 99%

“…We recently developed kisspeptin-based photoaffinity probes, where a photoreactive functional group was conjugated to potentially interactive residues in the sequence. 23 In this particular study, we used a panel of different probes to successfully identify several receptor binding residues for GPR54.…”

Section: Introductionmentioning

confidence: 99%

Design and synthesis of fluorescent probes for GPR54

Kaneda

Misu

Ohno

et al. 2014

Bioorganic & Medicinal Chemistry

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“…NMR studies using DPC micelles have shown that the C-terminal end of Kp1(10) forms a hydrophobic cluster consisting of Phe6 and Phe10 and the aliphatic chain of Leu8, which probably interacts with the carbon chains of the inner membrane phospholipids (Lee et al 2009a). The N-terminal region may also interact with the receptor as kisspeptin photoaffinity probe experiments with Kp1(54) and Kp1(14) show that the three N-terminal residues (Tyr, Asn, and Trp) can constitute a secondary binding site with KissR1 (Misu et al 2013).…”

Section: Interaction and Evolution Of Kiss/kissr Pairsmentioning

confidence: 99%

MOLECULAR EVOLUTION OF GPCRS: Kisspeptin/kisspeptin receptors

Pasquier¹,

Kamech²,

Lafont³

et al. 2014

Journal of Molecular Endocrinology

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Following the discovery of kisspeptin (Kiss) and its receptor (GPR54 or KissR) in mammals, phylogenetic studies revealed up to three Kiss and four KissR paralogous genes in other vertebrates. The multiplicity of Kiss and KissR types in vertebrates probably originated from the two rounds of whole-genome duplication (1R and 2R) that occurred in early vertebrates. This review examines compelling recent advances on molecular diversity and phylogenetic evolution of vertebrate Kiss and KissR. It also addresses, from an evolutionary point of view, the issues of the structure-activity relationships and interaction of Kiss with KissR and of their signaling pathways. Independent gene losses, during vertebrate evolution, have shaped the repertoire of Kiss and KissR in the extant vertebrate species. In particular, there is no conserved combination of a given Kiss type with a KissR type, across vertebrate evolution. The striking conservation of the biologically active ten-amino-acid C-terminal sequence of all vertebrate kisspeptins, probably allowed this evolutionary flexibility of Kiss/KissR pairs. KissR mutations, responsible for hypogonadotropic hypogonadism in humans, mostly occurred at highly conserved amino acid positions among vertebrate KissR. This further highlights the key role of these amino acids in KissR function. In contrast, less conserved KissR regions, notably in the intracellular C-terminal domain, may account for differential intracellular signaling pathways between vertebrate KissR. Cross talk between evolutionary and biomedical studies should contribute to further understanding of the Kiss/KissR structure-activity relationships and biological functions.

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“…However, each new unnatural amino acid that is addressed requires relatively large efforts of engineering of the critical RNA and protein components. In contrast, the cysteine/maleimide strategy used in this work has potential to be directly compatible with any new photoreactive probe produced as a maleimide derivative …”

Section: Discussionmentioning

confidence: 99%

Site‐Specific Photoconjugation of Beta‐Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split‐Enzyme Complementation

Alesand

Nygren

2018

Biotechnology Journal

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Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable.

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Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Cited by 9 publications

References 36 publications

Design and synthesis of fluorescent probes for GPR54

Design and synthesis of fluorescent probes for GPR54

MOLECULAR EVOLUTION OF GPCRS: Kisspeptin/kisspeptin receptors

Site‐Specific Photoconjugation of Beta‐Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split‐Enzyme Complementation

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