The Use of Bioluminescence Resonance Energy Transfer 2 to Study Neuropeptide Y Receptor Agonist-Induced β-Arrestin 2 Interaction

Berglund, Magnus; Schober, Douglas A.; Statnick, Michael A.; McDonald, Patricia; Gehlert, Donald R.

doi:10.1124/jpet.103.051227

Cited by 73 publications

(65 citation statements)

References 25 publications

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“…1229U91 has been suggested to be a partial agonist at Y4 receptors. Studies of agonist induced ␤-arrestin 2 translocation at the rhY4 receptor showed that the maximal response of hPP was 30% higher than that from 1229U91 (Berglund et al, 2003b). However, in the present study, the maximum and minimum levels were indistinguishable, consistent with full agonism for both peptides.…”

supporting

confidence: 52%

“…Our group has recently cloned the Y1, Y2, Y5 (Gehlert et al, 2001), and Y4 (Berglund et al, 2003b) receptors from the rhesus monkey. In this paper, we discuss the detailed characterization of the rhY4 receptor with regards to pharmacology and dimerization.…”

Section: Discussionmentioning

confidence: 99%

“…Based on the recently published sequence of the rhY4 receptor (Berglund et al, 2003b), one forward primer was synthesized: rhY4fH containing a HindIII site with the sequence 5Ј-AAGCTTAAGCTTACCATGAACACCTCT-CACCTCCT-3Ј and two reverse primers, rhY4rKS with the sequence 5Ј-CCGCGGTACCTTAAATGGGATTGGACCT-3Ј containing a KpnI and a SacI site and rhY4rNSKS (5Ј-CCGCGGTACCAATGGGATT-GGACCTGC-3Ј) also containing a KpnI and a SacI site but lacking the stop codon to make the carboxy terminally tagged constructs. The primer pair rhY4fH ϩ rhY4rKS were used in a PCR reaction using genomic rhesus DNA as template.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we cloned the rhY4 receptor and utilized BRET 2 to study agonist-induced ␤-arrestin 2 interaction in the NPY receptor family (Berglund et al, 2003b). In the present study, we explored the ability of the rhY4 receptor to form homoand/or heterodimers with the other members of the NPY receptor family.…”

mentioning

confidence: 99%

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Neuropeptide Y Y4 Receptor Homodimers Dissociate upon Agonist Stimulation

Berglund¹,

Schober²,

Esterman³

et al. 2003

J Pharmacol Exp Ther

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The pancreatic polypeptide-fold family of peptides consists of three 36-amino acid peptides, namely neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP). These peptides regulate important functions, including food intake, circadian rhythms, mood, blood pressure, intestinal secretion, and gut motility, through four receptors: Y1, Y2, Y4, and Y5. Additional receptor subtypes have been proposed based on pharmacology observed in native tissues. Recent studies with other Gprotein-coupled receptors have shown that homo-and heterodimerization may be important in determining receptor function and pharmacology. In the present study, the recently cloned rhesus (rh) Y4 receptor was evaluated using radioligand binding, and the pharmacological profile was found to be very similar to the human Y4 receptor. To study homo-and heterodimerization involving the Y4 receptor using bioluminescence resonance energy transfer 2 (BRET 2 ), the carboxy termini of the rhesus Y1, Y2, Y4, and Y5 receptors were fused to Renilla luciferase, and rhY4 was also fused to green fluorescent protein. Dimerization was also studied using Western blot analysis. Using both BRET 2 and Western analysis, we found that the rhY4 receptor is present at the cell surface as a homodimer. Furthermore, agonist stimulation using the Y4-selective agonists PP and 1229U91 can dissociate these dimers in a concentration-dependent manner. In contrast, rhY4 did not heterodimerize with other members of the NPY receptor family or with human opioid ␦ and receptors. Therefore, homodimerization is an important component in the regulation of the Y4 receptor.The neuropeptide Y (NPY) family of peptides consists of NPY, peptide YY (PYY), and pancreatic polypeptide (PP). These peptides regulate many important physiological functions, such as energy homeostasis, mood, and blood pressure. Currently, there are four cloned functional G-protein-coupled receptors (GPCRs), namely Y1, Y2, Y4, and Y5, that make up the NPY receptor family (see Berglund et al., 2003a for a review) in most mammals. All of these receptor subtypes are found in the brain as well as peripheral tissues. In addition, rabbits and mice have a functional y6 receptor, whereas in primates, this receptor subtype is not functional. NPY, PYY, and PP share a similar rank order of potencies at the Y1, Y2, and Y5 receptors: NPY Ϸ PYY Ͼ PP, whereas the Y4 receptor binds PP with higher affinity than it binds NPY and PYY and is thus regarded as the PP receptor. Like PP, the Y4 receptor is mainly found in the gut (Lundell et al., 1995), but binding sites for PP and Y4 mRNA have also been found in several rat brain regions, including hypothalamus and brainstem (Berglund et al., 2003a), suggesting that PP may also have direct effects on brain function. Besides the cloned NPY receptor family, several additional receptor subtypes have been proposed based on pharmacological evaluation of various tissue preparations. However, the first draft of the human genome did not provide evidence for any additional NPY receptor...

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supporting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Neuropeptide Y Y4 Receptor Homodimers Dissociate upon Agonist Stimulation

Berglund¹,

Schober²,

Esterman³

et al. 2003

J Pharmacol Exp Ther

Self Cite

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“…Currently, both BRET versions are being used for studying protein-protein interactions, including monitoring of GPCR activation by BRET-based β-arrestin recruitment assays. 15,[22][23][24][25][26][27][28][29] In this study, we report the development of an optimized BRET1-based β-arrestin2 assay for several GPCRs belonging to class A and class B receptors. Unlike most of the previous BRETbased β-arrestin studies that relied on the transient coexpression of the receptor and β-arrestin, the current work employed double stable cell lines that constitutively expressed optimal levels of both β-arrestin2-Rluc and GPCRs fused to a modified EYFP (VENUS), providing a robust and a mainstreamed platform for HTS of GPCR ligands.…”

mentioning

confidence: 99%

High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based β-Arrestin2 Recruitment Assay

Hamdan

Audet

Garneau³

et al. 2005

SLAS Discovery

167

143

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In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of β-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with β-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1-β-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and β-arrestin2-Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted β-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced β-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-β-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists. (Journal of Biomolecular Screening 2005:463-475)

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Peptide Modifications Differentially Alter G Protein‐Coupled Receptor Internalization and Signaling Bias

et al. 2014

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Although G protein‐coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti‐obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half‐lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.

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The Use of Bioluminescence Resonance Energy Transfer 2 to Study Neuropeptide Y Receptor Agonist-Induced β-Arrestin 2 Interaction

Cited by 73 publications

References 25 publications

Neuropeptide Y Y4 Receptor Homodimers Dissociate upon Agonist Stimulation

Neuropeptide Y Y4 Receptor Homodimers Dissociate upon Agonist Stimulation

High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based β-Arrestin2 Recruitment Assay

Peptide Modifications Differentially Alter G Protein‐Coupled Receptor Internalization and Signaling Bias

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