Structural Perspective on Ancient Neuropeptide Y-like System reveals Hallmark Features for Peptide Recognition and Receptor Activation

Gershkovich, Miron Mikhailowitsch; Groß, Victoria Elisabeth; Vu, Oanh; Schoeder, Clara T.; Meiler, Jens; Prömel, Simone; Kaiser, Anette

doi:10.1016/j.jmb.2021.166992

Cited by 6 publications

(8 citation statements)

References 55 publications

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“…S 3 A). These data are consistent with previously performed competition-binding assays using TAM-FLP-34-1 as fluorescent tracer, in which unmodified FLP-34-1 displays an inhibition binding constant K i of 107 nM [ 35 ]. This is comparable to the K d value of the fluorescently labeled peptide, and thus, corroborates the wild type-like properties of TAM-FLP-34-1.…”

Section: Resultssupporting

confidence: 91%

“…For NanoBRET binding assays, the nanoluciferase (Nluc, Promega, USA) was genetically fused to the N terminus of npr-11 , spaced by a SGGGGS linker as previously described [ 35 ]. To facilitate expression and targeting to the plasma membrane, the Nluc sequence was preceded by a secretion signal derived from human IL-6 as previously described (secNluc [ 5 ];).…”

Section: Methodsmentioning

confidence: 99%

“…The binding of TAM-FLP-34-1 and its scrambled analog was also tested in vitro using membranes of transfected HEK293 cells as described previously [ 35 ]. Briefly, membranes of HEK293 cells transiently transfected with Nluc::NPR-11::eYFP were prepared analogously to a described protocol [ 67 , 68 ].…”

Section: Methodsmentioning

confidence: 99%

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NanoBRET in C. elegans illuminates functional receptor interactions in real time

Groß

Gershkovich

Schöneberg

et al. 2022

BMC Mol and Cell Biol

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Background Protein-protein interactions form the basis of every organism and thus, investigating their dynamics, intracellular protein localization, trafficking and interactions of distinct proteins such as receptors and their ligand-binding are of general interest. Bioluminescence resonance energy transfer (BRET) is a powerful tool to investigate these aspects in vitro. Since in vitro approaches mostly neglect the more complex in vivo situation, we established BRET as an in vivo tool for studying protein interactions in the nematode C. elegans. Results We generated worms expressing NanoBRET sensors and elucidated the interaction of two ligand-G protein-coupled receptor (GPCR) pairs, the neuropeptide receptor NPR-11 and the Adhesion GPCR LAT-1. Furthermore, we adapted the enhanced bystander BRET technology to measure subcellular protein localization. Using this approach, we traced ligand-induced internalization of NPR-11 in vivo. Conclusions Our results indicate that in vivo NanoBRET is a tool to investigate specific protein interactions and localization in a physiological setting in real time in the living organism C. elegans.

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Section: Resultssupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

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NanoBRET in C. elegans illuminates functional receptor interactions in real time

Groß

Gershkovich

Schöneberg

et al. 2022

BMC Mol and Cell Biol

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“…5, A to C). These three residues are highly conserved in all NPY receptors throughout evolution ( 42 , 43 ), suggesting that they may have similar key functions in all NPY receptor subtypes. Previous characterization of modifications of the amide group at the peptide C terminus showed a marked loss of binding affinity to Y 1 R, Y 2 R, and Y 4 R ( 38 , 44 , 45 ), underlining a crucial role of the amide in binding to the NPY receptors.…”

Section: Resultsmentioning

confidence: 99%

Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors

et al. 2022

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In response to three highly conserved neuropeptides, neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP), four G protein–coupled receptors mediate multiple essential physiological processes, such as food intake, vasoconstriction, sedation, and memory retention. Here, we report the structures of the human Y 1 , Y 2 , and Y 4 receptors in complex with NPY or PP, and the G i1 protein. These structures reveal distinct binding poses of the peptide upon coupling to different receptors, reflecting the importance of the conformational plasticity of the peptide in recognizing the NPY receptors. The N terminus of the peptide forms extensive interactions with the Y 1 receptor, but not with the Y 2 and Y 4 receptors. Supported by mutagenesis and functional studies, subtype-specific interactions between the receptors and peptides were further observed. These findings provide insight into key factors that govern NPY signal recognition and transduction, and would enable development of selective drugs.

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“…The investigation of direct ligand/receptor interactions was performed by double-mutant cycle analysis. [24] This is a powerful technique as demonstrated to identify critical amino acid residues for the NPR-1 receptor [36] or the neuropeptide Y receptors. [37] For chemerin, R 94 , K 95 , R 96 , K 97 and H 38 , K 39 , H 40 , R 78 , K 79 , R 80 were exchanged to alanine in clusters.…”

Section: Discussionmentioning

confidence: 99%

Stable Binding of Full‐Length Chemerin Is Driven by Negative Charges in the CMKLR1 N Terminus

et al. 2023

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The adipokine chemerin is the endogenous ligand of the chemokine‐like receptor 1 (CMKLR1), a member of the family of G protein‐coupled receptors (GPCRs). This protein ligand plays an important role in obesity and inflammatory processes. Stable receptor–ligand interactions are highly relevant for its different physiological effects such as the migration of immune cells towards sites of inflammation. Here, we demonstrate that negative charges in the CMKLR1 N terminus are involved in the formation of strong contacts with a specific positively charged patch at the surface of full‐length chemerin, which is absent in the short nonapeptide agonist chemerin‐9, thus explaining its reduced affinity. Using receptor chimera of G protein‐coupled receptor 1 (GPR1) and CMKLR1, we were able to identify the residues of this interaction and its relevance for stable full‐length chemerin binding. This could help to develop more potent ligands for the treatment of inflammation‐related diseases.

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Structural Perspective on Ancient Neuropeptide Y-like System reveals Hallmark Features for Peptide Recognition and Receptor Activation

Cited by 6 publications

References 55 publications

NanoBRET in C. elegans illuminates functional receptor interactions in real time

NanoBRET in C. elegans illuminates functional receptor interactions in real time

Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors

Stable Binding of Full‐Length Chemerin Is Driven by Negative Charges in the CMKLR1 N Terminus

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