[Leu31-Pro34] neuropeptide Y identifies a subtype of 125I-labeled peptide YY binding sites in the rat brain

Gehlert, Donald R.; Gackenheimer, Susan L.; Schober, Douglas A.

doi:10.1016/0197-0186(92)90067-2

Cited by 67 publications

(43 citation statements)

References 76 publications

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“…Pellets of Rluc-tagged rhY1-, rhY2-, and rhY4-expressing cells were frozen as aliquots at Ϫ80°C, whereas rhY5-Rluc membranes were used immediately to avoid receptor degradation. Radioligand binding assays were conducted on isolated crude membrane homogenates as described previously (Gehlert et al, 1992) using iodinated human (h) PYY (Y1, Y2, and Y5) or hPP (Y4) as radioligands. Nonspecific binding was defined as the amount of radioactivity remaining on the filter after incubating in the presence of 0.1 M hPYY (American Peptide Co., Inc., Sunnyvale, CA) for Y1, Y2, and Y5 or 0.1 M hPP for Y4 (American Peptide Co., Inc.).…”

Section: Methodsmentioning

confidence: 99%

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

Berglund¹,

Schober²,

Statnick³

et al. 2003

J Pharmacol Exp Ther

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The neuropeptide Y (NPY) family peptides NPY, peptide YY (PYY), and pancreatic polypeptide (PP) bind to four G proteincoupled receptors (GPCRs): Y1, Y2, Y4, and Y5. A key step in the desensitization and internalization of GPCRs is the association of the receptor with ␤-arrestins. In the present study, these receptors were analyzed with respect to their ability to interact with GFP2-tagged ␤-arrestin 2 using the new bioluminescence resonance energy transfer 2 method. Agonists induced a concentration-dependent association of ␤-arrestin 2 with all four receptors. Whereas the Y1 receptor exhibited the highest maximum response and rapid association (t 1/2 ϭ 3.4 min), the maximal signals for the association of Y2 and Y4 receptors were less than half of that of Y1, and the association rates were much slower. Interestingly, when evaluated at the Y4 receptor, the Y4 agonist 1229U91 [(Ile,Glu,Pro,Dpr,Tyr,Arg, Leu,Arg,Try-NH 2 )-2-cyclic(2,4Ј),(2Ј,4)-diamide] was unable to provoke the same maximal response as human PP, suggesting that 1229U91 is a partial agonist. When stimulated by PYY, the Y5 receptor responded with a t 1/2 of 4.6 min and a maximal response approximately 60% of what was observed with Y1. Because ␤-arrestins are key components in GPCR internalization, it is interesting to note that the receptor that is known to internalize rapidly (Y1) exhibits the most rapid association with ␤-arrestin 2, whereas the receptor that is known to internalize slowly, or not at all (Y2) associates slowly with ␤-arrestin 2.

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Section: Methodsmentioning

confidence: 99%

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

Berglund¹,

Schober²,

Statnick³

et al. 2003

J Pharmacol Exp Ther

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“…HEK293 cells stably expressing the rhesus Y4 (WT) receptor and a cell line expressing the recombinant rhesus Y4-GFP 2 receptor protein were washed once with phosphatebuffered saline (PBS) and pelleted in fresh PBS. Radioligand binding assays were conducted on isolated crude membrane homogenates as previously described (Gehlert et al, 1992) using 125 I-hPP as radioligand. Nonspecific binding was defined as the amount of radioactivity remaining on the filter after incubating in the presence of 0.1 M human (h)PP (American Peptide Co., Inc., Sunnyvale, CA).…”

Section: Methodsmentioning

confidence: 99%

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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“…Several studies using [ 125 I]‐NPY or [ 125 I]‐PYY as radioligands have demonstrated that the rat brain contained high amounts of NPY receptors distributed in various brain structures including the cortex, the hippocampus, multiple thalamic, hypothalamic and brainstem nuclei, and the cerebellum and the spinal cord (Dumont et al ., 1992; Lynch et al ., 1989; Martel et al ., 1990; Kar & Quirion, 1992; Quirion & Martel, 1992). Subsequent autoradiograph studies using [ 125 I]‐PYY in the presence of [Pro 34 ]‐PYY or C‐terminal NPY/PYY fragments revealed the existence of at least two classes (Y 1 ‐like and Y 2 ‐like) of receptors in the rat brain (Aicher et al ., 1991; Dumont et al ., 1990; 1993; Gehlert et al ., 1992; Larsen et al ., 1993). The differential distribution of these receptor subtypes was confirmed using more selective radioligands such as [ 125 I]‐Leu 31 ,Pro 34 ]‐PYY (Y 1 ‐like) and [ 125 I]‐PYY 3–36 (Y 2 ‐like) (Dumont et al ., 1995; 1996a; Gehlert & Gackenheimer, 1997).…”

Section: Introductionmentioning

confidence: 99%

[¹²⁵I]‐GR231118: a high affinity radioligand to investigate neuropeptide Y Y₁ and Y₄ receptors

Dumont

Quirion

2000

British J Pharmacology

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GR231118 (also known as 1229U91 and GW1229), a purported Y1 antagonist and Y4 agonist was radiolabelled using the chloramine T method. [125I]‐GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [125I]‐GR231118 binds with very high affinity (Kd of 0.09–0.24 nM) in transfected HEK293 cells with the rat Y1 and Y4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y2 or Y5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [125I]‐GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y1, but not rat Y4, receptor cDNA. Autoradiographic studies demonstrated that [125I]‐GR231118 binding sites were fully inhibited by the Y1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [125I]‐GR231118/BIBO3304‐insensitive binding sites were detected in few areas. These [125I]‐GR231118/BIBO3304‐insensitive binding sites likely represent labelling to the Y4 receptor subtype. In summary, [125I]‐GR231118 is a new radiolabelled probe to investigate the Y1 and Y4 receptors; its major advantage being its high affinity. Using highly selective Y1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [125I]‐GR231118 to the Y1 receptor allowing for the characterization and visualization of the purported Y4 subtype. British Journal of Pharmacology (2000) 129, 37–46; doi:

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[Leu31-Pro34] neuropeptide Y identifies a subtype of 125I-labeled peptide YY binding sites in the rat brain

Cited by 67 publications

References 76 publications

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

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

Neuropeptide Y Y4 Receptor Homodimers Dissociate upon Agonist Stimulation

[¹²⁵I]‐GR231118: a high affinity radioligand to investigate neuropeptide Y Y₁ and Y₄ receptors

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[Leu31-Pro34] neuropeptide Y identifies a subtype of 125I-labeled peptide YY binding sites in the rat brain

Cited by 67 publications

References 76 publications

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

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

Neuropeptide Y Y4 Receptor Homodimers Dissociate upon Agonist Stimulation

[125I]‐GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors

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[¹²⁵I]‐GR231118: a high affinity radioligand to investigate neuropeptide Y Y₁ and Y₄ receptors